Structured Review

Waters Corporation rapigest
Workflow for large-scale quantitative phosphoproteomics of synaptosomes. Synaptosomal proteins were extracted by lysis of synaptosomes and acetone precipitation. The protein pellets were dried, resuspended in 1% <t>RapiGest</t> buffer and digested by trypsin. Peptides were labeled with heavy and light dimethyl labeling ( Boersema et al., 2009 ). To obtain pair-wise comparisons between the three conditions, samples were mixed with a ratio of 1:1. The mixed peptides were separated by strong cation exchange (SCX) chromatography, with the eluate being divided into 12 fractions. Phosphopeptides in each fraction were separately enriched by TiO 2 microbeads ( Larsen et al., 2005 ) and subjected for mass spectrometry analysis. DOI: http://dx.doi.org/10.7554/eLife.14530.005
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Images

1) Product Images from "Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis"

Article Title: Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis

Journal: eLife

doi: 10.7554/eLife.14530

Workflow for large-scale quantitative phosphoproteomics of synaptosomes. Synaptosomal proteins were extracted by lysis of synaptosomes and acetone precipitation. The protein pellets were dried, resuspended in 1% RapiGest buffer and digested by trypsin. Peptides were labeled with heavy and light dimethyl labeling ( Boersema et al., 2009 ). To obtain pair-wise comparisons between the three conditions, samples were mixed with a ratio of 1:1. The mixed peptides were separated by strong cation exchange (SCX) chromatography, with the eluate being divided into 12 fractions. Phosphopeptides in each fraction were separately enriched by TiO 2 microbeads ( Larsen et al., 2005 ) and subjected for mass spectrometry analysis. DOI: http://dx.doi.org/10.7554/eLife.14530.005
Figure Legend Snippet: Workflow for large-scale quantitative phosphoproteomics of synaptosomes. Synaptosomal proteins were extracted by lysis of synaptosomes and acetone precipitation. The protein pellets were dried, resuspended in 1% RapiGest buffer and digested by trypsin. Peptides were labeled with heavy and light dimethyl labeling ( Boersema et al., 2009 ). To obtain pair-wise comparisons between the three conditions, samples were mixed with a ratio of 1:1. The mixed peptides were separated by strong cation exchange (SCX) chromatography, with the eluate being divided into 12 fractions. Phosphopeptides in each fraction were separately enriched by TiO 2 microbeads ( Larsen et al., 2005 ) and subjected for mass spectrometry analysis. DOI: http://dx.doi.org/10.7554/eLife.14530.005

Techniques Used: Lysis, Labeling, Chromatography, Mass Spectrometry

2) Product Images from "Affinity pulldown of γ-secretase and associated proteins from human and rat brain"

Article Title: Affinity pulldown of γ-secretase and associated proteins from human and rat brain

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/j.1582-4934.2009.00907.x

γ-Secretase complex components were specifically captured by GCB pulldown from human frozen brain material. Solubilized γ-secretase prepared from human brain (frontal cortex) was incubated with 200 nM GCB in the presence (+) or the absence (−) of 10 μM L-685,458 and isolated with SA beads. The captured γ-secretase complex was eluted by 10 mM DTT supplement with 0.01% RapiGest and subjected to Western blotting for the indicated γ-secretase subunit. The density of the bands was calculated as a percentage of a standard (input sample) run on the same gel.
Figure Legend Snippet: γ-Secretase complex components were specifically captured by GCB pulldown from human frozen brain material. Solubilized γ-secretase prepared from human brain (frontal cortex) was incubated with 200 nM GCB in the presence (+) or the absence (−) of 10 μM L-685,458 and isolated with SA beads. The captured γ-secretase complex was eluted by 10 mM DTT supplement with 0.01% RapiGest and subjected to Western blotting for the indicated γ-secretase subunit. The density of the bands was calculated as a percentage of a standard (input sample) run on the same gel.

Techniques Used: Incubation, Isolation, Western Blot

3) Product Images from "The beauty of being (label)-free: sample preparation methods for SWATH-MS and next-generation targeted proteomics"

Article Title: The beauty of being (label)-free: sample preparation methods for SWATH-MS and next-generation targeted proteomics

Journal: F1000Research

doi: 10.12688/f1000research.2-272.v2

In solution digestion leads to stable results in label-free proteomics. ( a ) The distribution maximum of coefficients of variation (CV) of the selected protocols varies between 0.075 and 0.2. CV values obtained for protocol triplicates are shown as two-dimensional distribution histograms (‘violin plots’). Quantification in DDA experiments was consistent over the dynamic range, as CV values only marginally changed when filtering by peptides according to their abundance (80%, 60%, 40% or 20%). CV likelihood maxima of all protocols were below 20%, while RapidACN led to the most reproducible results (CV = 7%) (n = 3). ( b ) Stability in a quantification experiment is improved by data-independent acquisition. CV values for the same set of peptides measured with SWATH and DDA using the RapiGest protocol, as shown in a two-dimensional distribution histogram. Whereas there was a high signal variation in DDA acquisition, the variation could be largely reduced in SWATH acquisition. ( c ) In solution protocols yield the highest number of peptides suitable for label-free quantification. The number of peptides with a CV
Figure Legend Snippet: In solution digestion leads to stable results in label-free proteomics. ( a ) The distribution maximum of coefficients of variation (CV) of the selected protocols varies between 0.075 and 0.2. CV values obtained for protocol triplicates are shown as two-dimensional distribution histograms (‘violin plots’). Quantification in DDA experiments was consistent over the dynamic range, as CV values only marginally changed when filtering by peptides according to their abundance (80%, 60%, 40% or 20%). CV likelihood maxima of all protocols were below 20%, while RapidACN led to the most reproducible results (CV = 7%) (n = 3). ( b ) Stability in a quantification experiment is improved by data-independent acquisition. CV values for the same set of peptides measured with SWATH and DDA using the RapiGest protocol, as shown in a two-dimensional distribution histogram. Whereas there was a high signal variation in DDA acquisition, the variation could be largely reduced in SWATH acquisition. ( c ) In solution protocols yield the highest number of peptides suitable for label-free quantification. The number of peptides with a CV

Techniques Used:

Protocols cover cellular compartments differently. ( a ) RapiGest and eFASP cover a unique space in the proteome. Identified proteins were visualised in a Venn diagram, excluding the in gel protocols. The RapiGest procedure yielded most unique IDs, followed by eFASP and RapidACN (n = 3). ( b ) SDS-containing protocols are best suited for the extraction of membrane proteins. For the analysis of annotated functions in each protocol, selected GO terms were expressed as percentages of identified proteins. While cytosolic proteins were not enriched in any protocol, membrane proteins were preferentially detected in the SDS-containing protocols. (n = 3, Error bars = +/- S.D.) ( c ) Filter-aided sample preparations yield a balanced representation of the proteome. The identified proteins were plotted against the percentage of proteins annotated by the GO term cytosol, in order to illustrate the similarity of extraction properties. The protocol properties required for efficient extraction of membrane and nuclear proteins is inversely correlated with the extraction efficiency for cytosolic proteins, while there is a positive correlation with ribosomal proteins.
Figure Legend Snippet: Protocols cover cellular compartments differently. ( a ) RapiGest and eFASP cover a unique space in the proteome. Identified proteins were visualised in a Venn diagram, excluding the in gel protocols. The RapiGest procedure yielded most unique IDs, followed by eFASP and RapidACN (n = 3). ( b ) SDS-containing protocols are best suited for the extraction of membrane proteins. For the analysis of annotated functions in each protocol, selected GO terms were expressed as percentages of identified proteins. While cytosolic proteins were not enriched in any protocol, membrane proteins were preferentially detected in the SDS-containing protocols. (n = 3, Error bars = +/- S.D.) ( c ) Filter-aided sample preparations yield a balanced representation of the proteome. The identified proteins were plotted against the percentage of proteins annotated by the GO term cytosol, in order to illustrate the similarity of extraction properties. The protocol properties required for efficient extraction of membrane and nuclear proteins is inversely correlated with the extraction efficiency for cytosolic proteins, while there is a positive correlation with ribosomal proteins.

Techniques Used:

Protein identification in label-free sample preparations. ( a ) Proteolytic digestion efficiencies. Trypsin or Lys-C/trypsin (FASP) digestion efficiencies expressed as relative occurrence of spectra that could be assigned miscleaved peptides (n = 3). ( b ) Amino acid specificity of proteolytic digestion. Relative occurrence of identified peptides with C-terminal lysine or arginine, compared to the average frequency of these amino acids across all individual proteins identified (n = 3, Error bars = +/- S.D.) ( c ) Identified peptides differ per protocol, and correlate with the total peak area as recorded in a DDA experiment. 18 samples derived from the same yeast culture were processed with six protocols in triplicates, and analyzed on a TripleTOF5600 instrument. The number of identified peptides correlates with the total peak area recorded, and indicates the highest identification rate in in solution digests, followed by filter-aided , and in gel procedures. ( d ) Detection of proteins by DDA or SWATH in a label-free experiment. Samples were analyzed in triplicates both for DDA and SWATH acquisition on a TripleTOF5600 instrument, data was searched using paragon (DDA), and Spectronaut (SWATH). SWATH increased the number of detectable proteins in combination with the in solution protocols. In solution protocols RapidACN and RapiGest led to the detection of up to 1000 proteins in single injections, followed by FASP and eFASP, which gave rise to between 250 and 750 proteins, and in gel injections that yielded 300 proteins IDs. Inset: A comparison of protein IDs from the TripleTOF and QExactive platforms shows a linear correlation for the protocols investigated. Data was searched using Mascot (n = 3, Error bars = +/- S.D.).
Figure Legend Snippet: Protein identification in label-free sample preparations. ( a ) Proteolytic digestion efficiencies. Trypsin or Lys-C/trypsin (FASP) digestion efficiencies expressed as relative occurrence of spectra that could be assigned miscleaved peptides (n = 3). ( b ) Amino acid specificity of proteolytic digestion. Relative occurrence of identified peptides with C-terminal lysine or arginine, compared to the average frequency of these amino acids across all individual proteins identified (n = 3, Error bars = +/- S.D.) ( c ) Identified peptides differ per protocol, and correlate with the total peak area as recorded in a DDA experiment. 18 samples derived from the same yeast culture were processed with six protocols in triplicates, and analyzed on a TripleTOF5600 instrument. The number of identified peptides correlates with the total peak area recorded, and indicates the highest identification rate in in solution digests, followed by filter-aided , and in gel procedures. ( d ) Detection of proteins by DDA or SWATH in a label-free experiment. Samples were analyzed in triplicates both for DDA and SWATH acquisition on a TripleTOF5600 instrument, data was searched using paragon (DDA), and Spectronaut (SWATH). SWATH increased the number of detectable proteins in combination with the in solution protocols. In solution protocols RapidACN and RapiGest led to the detection of up to 1000 proteins in single injections, followed by FASP and eFASP, which gave rise to between 250 and 750 proteins, and in gel injections that yielded 300 proteins IDs. Inset: A comparison of protein IDs from the TripleTOF and QExactive platforms shows a linear correlation for the protocols investigated. Data was searched using Mascot (n = 3, Error bars = +/- S.D.).

Techniques Used: Derivative Assay

Characteristics of label-free sample preparation methods. Left panel: Schematic overview of the different steps in an LC-MS/MS sample preparation method. Right panel: Main characteristics of the protocols compared in this study. Detailled protocols are given in the Supplementary material . Supplementary protocol 1 : In gel/SDS; 2: In gel/ABC; 3: FASP; 4: eFASP; 5: RapiGest; 6: RapidACN.
Figure Legend Snippet: Characteristics of label-free sample preparation methods. Left panel: Schematic overview of the different steps in an LC-MS/MS sample preparation method. Right panel: Main characteristics of the protocols compared in this study. Detailled protocols are given in the Supplementary material . Supplementary protocol 1 : In gel/SDS; 2: In gel/ABC; 3: FASP; 4: eFASP; 5: RapiGest; 6: RapidACN.

Techniques Used: Sample Prep, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

Bias of protein size and pI in sample preparation. ( a ) The spectrum of protein sizes is well covered for all protocols examined. The number of identified proteins was plotted against the theoretical molecular weight (MW) for each protocol investigated. Although some protocols yielded higher identifications than others, the MW range was highly reproducible for all of them when comparing against the whole yeast proteome. ( b ) Different representations of protein charges. When comparing to the distribution of theoretical protein pI values of the whole proteome, all investigated protocols showed an under-representation of proteins with a pI of 10. When expressing the total deviation as deviation score d, RapiGest, FASP and RapidACN score best. The d values were calculated as the sum of all differences in % compared to the theoretical proteome occurrence multiplied by 0.1.
Figure Legend Snippet: Bias of protein size and pI in sample preparation. ( a ) The spectrum of protein sizes is well covered for all protocols examined. The number of identified proteins was plotted against the theoretical molecular weight (MW) for each protocol investigated. Although some protocols yielded higher identifications than others, the MW range was highly reproducible for all of them when comparing against the whole yeast proteome. ( b ) Different representations of protein charges. When comparing to the distribution of theoretical protein pI values of the whole proteome, all investigated protocols showed an under-representation of proteins with a pI of 10. When expressing the total deviation as deviation score d, RapiGest, FASP and RapidACN score best. The d values were calculated as the sum of all differences in % compared to the theoretical proteome occurrence multiplied by 0.1.

Techniques Used: Sample Prep, Molecular Weight, Expressing

4) Product Images from "Improved protease digestion conditions for membrane protein detection"

Article Title: Improved protease digestion conditions for membrane protein detection

Journal: Electrophoresis

doi: 10.1002/elps.201400579

E. coli proteomic study. (4A, 4B) Unique proteins and membrane proteins identified from the membrane protein fraction (MPF) under standard digestion condition, SDS, and MeOH/RapiGest with trypsin and chymotrypsin digestions; in 4B, fractions above the
Figure Legend Snippet: E. coli proteomic study. (4A, 4B) Unique proteins and membrane proteins identified from the membrane protein fraction (MPF) under standard digestion condition, SDS, and MeOH/RapiGest with trypsin and chymotrypsin digestions; in 4B, fractions above the

Techniques Used:

Effects of additives on protease activity. (1A) Effects of MeOH; (1B) Effects of SDS and RapiGest.
Figure Legend Snippet: Effects of additives on protease activity. (1A) Effects of MeOH; (1B) Effects of SDS and RapiGest.

Techniques Used: Activity Assay

5) Product Images from "Proteomic Analysis of the Extracellular Matrix Produced by Mesenchymal Stromal Cells: Implications for Cell Therapy Mechanism"

Article Title: Proteomic Analysis of the Extracellular Matrix Produced by Mesenchymal Stromal Cells: Implications for Cell Therapy Mechanism

Journal: PLoS ONE

doi: 10.1371/journal.pone.0079283

Proteins that exhibit differential expression between SB623- and MSC-derived ECM; either in SDS/urea-soluble or SDS/urea-insoluble fractions or both. Precipitated ECM samples were resuspended in 0.1% w/v ammonium bicarbonate and Rapigest surfactant powder. The sample was then reduced, alkylated, trypsinized and analyzed by shotgun nLC-MS/MS. Proteins that were significantly different in abundance between SB623- and corresponding MSC-ECM are plotted in (A), with the exception of fibronectin (FN1), which is plotted in (B) because of its relatively high abundance. Collagen 1 alpha 1 (COL1A1), collagen 6 alpha 1 (COL6A1), collagen 6 alpha 3 (COL6A3), perlecan (HSPG2), latent transforming growth factor binding protein 1 (LTBP1), tenascin-c (TNC), transforming growth factor-beta-induced protein ig-h3 (TGFBI), transglutaminase 2 (TGM2). Mean ± SD; *p-value
Figure Legend Snippet: Proteins that exhibit differential expression between SB623- and MSC-derived ECM; either in SDS/urea-soluble or SDS/urea-insoluble fractions or both. Precipitated ECM samples were resuspended in 0.1% w/v ammonium bicarbonate and Rapigest surfactant powder. The sample was then reduced, alkylated, trypsinized and analyzed by shotgun nLC-MS/MS. Proteins that were significantly different in abundance between SB623- and corresponding MSC-ECM are plotted in (A), with the exception of fibronectin (FN1), which is plotted in (B) because of its relatively high abundance. Collagen 1 alpha 1 (COL1A1), collagen 6 alpha 1 (COL6A1), collagen 6 alpha 3 (COL6A3), perlecan (HSPG2), latent transforming growth factor binding protein 1 (LTBP1), tenascin-c (TNC), transforming growth factor-beta-induced protein ig-h3 (TGFBI), transglutaminase 2 (TGM2). Mean ± SD; *p-value

Techniques Used: Expressing, Derivative Assay, Mass Spectrometry, Binding Assay

6) Product Images from "Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65"

Article Title: Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65

Journal: Biochemical Journal

doi: 10.1042/BJ20140334

Identification of a highly conserved ubiquitin phospho-Ser 65 peptide upon PINK1 stimulation by CCCP in vivo New highly conserved ubiquitin phospho-Ser 65 peptide is up-regulated upon cell treatment with CCCP. Flp-In T-Rex HEK-293 cells stably expressing FLAG-empty, wild-type PINK1–FLAG or kinase-inactive PINK1–FLAG were grown in light, heavy and medium SILAC media respectively. Cells under each condition were stimulated with 10 μM CCCP for 3 h. Subsequently, membrane fractions were enriched by ultracentrifugation and solubilized in 1% RapiGest. Lysates from each of the three conditions were mixed at 1:1:1 and digested with trypsin before phosphopeptide enrichment by HILIC (hydrophilic-interaction LC) and TiO 2 , and analysis by MS. Data analysis was performed using MaxQuant. The experiment was performed using four replicates. ( A ) Representative extracted ion chromatograms representing the ubiquitin Ser 65 ) phosphopeptide TLSDYNIQKEpSTLHLVLR in the three SILAC-labelled conditions. ( B ) Sequence alignment of residues around Ser 65 in human Parkin and ubiquitin in a variety of organisms showing a high degree of conservation. C. elegans , Caenorhabditis elegans ; D. melanogaster , Drosophila melanogaster ; H. sapiens , Homo sapiens ; M. musculus , Mus musculus ; S. cerevisiae , Saccharomyces cerevisiae .
Figure Legend Snippet: Identification of a highly conserved ubiquitin phospho-Ser 65 peptide upon PINK1 stimulation by CCCP in vivo New highly conserved ubiquitin phospho-Ser 65 peptide is up-regulated upon cell treatment with CCCP. Flp-In T-Rex HEK-293 cells stably expressing FLAG-empty, wild-type PINK1–FLAG or kinase-inactive PINK1–FLAG were grown in light, heavy and medium SILAC media respectively. Cells under each condition were stimulated with 10 μM CCCP for 3 h. Subsequently, membrane fractions were enriched by ultracentrifugation and solubilized in 1% RapiGest. Lysates from each of the three conditions were mixed at 1:1:1 and digested with trypsin before phosphopeptide enrichment by HILIC (hydrophilic-interaction LC) and TiO 2 , and analysis by MS. Data analysis was performed using MaxQuant. The experiment was performed using four replicates. ( A ) Representative extracted ion chromatograms representing the ubiquitin Ser 65 ) phosphopeptide TLSDYNIQKEpSTLHLVLR in the three SILAC-labelled conditions. ( B ) Sequence alignment of residues around Ser 65 in human Parkin and ubiquitin in a variety of organisms showing a high degree of conservation. C. elegans , Caenorhabditis elegans ; D. melanogaster , Drosophila melanogaster ; H. sapiens , Homo sapiens ; M. musculus , Mus musculus ; S. cerevisiae , Saccharomyces cerevisiae .

Techniques Used: In Vivo, Stable Transfection, Expressing, Hydrophilic Interaction Liquid Chromatography, Mass Spectrometry, Sequencing

7) Product Images from "mTORC1 promotes T-bet phosphorylation to regulate Th1 differentiation"

Article Title: mTORC1 promotes T-bet phosphorylation to regulate Th1 differentiation

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1601078

Three of the novel T-bet phosphorylations are mTORC1 dependent A, Wild-type C57/BL6 (WT) or Rheb fl/fl CD4+ Cre (Rheb KO) CD4+ T cells were purified from spleens and lymph nodes of 6–12 week old mice and activated with plate-bound anti-CD3 and soluble anti-CD28 for 24 hours in Th1 polarizing culture conditions. Activated T cells were harvested, lysed with Rapigest, trypsin digested and analyzed by LC-MRM-MS. AUC on the y axis is area under the curve – measure of phosphorylated peptide abundance. Data are from one of 2 experiments with T cells from individual mice. B, CD4+ T cells were purified from spleens and lymph nodes of WT mice and activated for 48 hours as in A in the presence or absence of 500 nM Rapamycin (WT or WT Rapamycin), prepared and analyzed as in A. Data are from one of 3 experiments with T cells from individual mice. C, Schematic of T-bet with phosphorylations shown as circles with numbers. Gray circles are the phosphorylations sites. White circles show phosphorylations that are mTORC1 dependent. Data are from one of 5 experiments with T cells from individual mice.
Figure Legend Snippet: Three of the novel T-bet phosphorylations are mTORC1 dependent A, Wild-type C57/BL6 (WT) or Rheb fl/fl CD4+ Cre (Rheb KO) CD4+ T cells were purified from spleens and lymph nodes of 6–12 week old mice and activated with plate-bound anti-CD3 and soluble anti-CD28 for 24 hours in Th1 polarizing culture conditions. Activated T cells were harvested, lysed with Rapigest, trypsin digested and analyzed by LC-MRM-MS. AUC on the y axis is area under the curve – measure of phosphorylated peptide abundance. Data are from one of 2 experiments with T cells from individual mice. B, CD4+ T cells were purified from spleens and lymph nodes of WT mice and activated for 48 hours as in A in the presence or absence of 500 nM Rapamycin (WT or WT Rapamycin), prepared and analyzed as in A. Data are from one of 3 experiments with T cells from individual mice. C, Schematic of T-bet with phosphorylations shown as circles with numbers. Gray circles are the phosphorylations sites. White circles show phosphorylations that are mTORC1 dependent. Data are from one of 5 experiments with T cells from individual mice.

Techniques Used: Purification, Mouse Assay, Mass Spectrometry

8) Product Images from "Mechanism-based Inhibition of iPLA2β Demonstrates a Highly Reactive Cysteine Residue (C651) That Interacts with the Active Site: Mass Spectrometric Elucidation of the Mechanisms of Underlying Inhibition"

Article Title: Mechanism-based Inhibition of iPLA2β Demonstrates a Highly Reactive Cysteine Residue (C651) That Interacts with the Active Site: Mass Spectrometric Elucidation of the Mechanisms of Underlying Inhibition

Journal: Biochemistry

doi: 10.1021/bi4004233

Comparison of the extent of iPLA 2 β modification by (S) -BEL and (R) -BEL An equal amount of purified iPLA 2 β was incubated with either (S) -BEL or (R) -BEL at a molar ratio of 1:1 at 22°C for 3 min. The reaction was terminated by precipitating the protein with chloroform/methanol. The protein pellet was solubilized in buffer containing RapiGest ™ , digested with trypsin, and analyzed by LC/MS/MS as described in “Experimental Procedures”. The normalized relative abundance of a target peptide was calculated as described in “Experimental Procedures”. A , the normalized relative abundance of the BEL-modified S465-containing tryptic peptide (D456-K478) in (R) -BEL- or (S) -BEL-treated samples. B , the normalized relative abundance of the BEL modified C651-containing tryptic peptide (S644-K665) in (R) -BEL- or (S) -BEL-treated samples. C , the normalized relative abundance of the cross-linked peptide in (R) -BEL or (S) -BEL-treated samples. The results indicate that treatment with (S) -BEL results in a higher level of protein modification. By examining the changes in band intensity following SDS-PAGE in the time-course experiment and comparing the levels of modified S465 and C651 as determined by ESI-LC/MS/MS, we found that (S) -BEL is more efficient at covalent modification of iPLA 2 β than (R) -BEL. These observations are in good agreement with the fact that (S) -BEL is a selective inhibitor of iPLA 2 β, and demonstrate the enantioselectivity of (S) - vs. (R)- BEL in the kinetics of covalent modification of iPLA 2 β leading to inhibition of enzymic activity.
Figure Legend Snippet: Comparison of the extent of iPLA 2 β modification by (S) -BEL and (R) -BEL An equal amount of purified iPLA 2 β was incubated with either (S) -BEL or (R) -BEL at a molar ratio of 1:1 at 22°C for 3 min. The reaction was terminated by precipitating the protein with chloroform/methanol. The protein pellet was solubilized in buffer containing RapiGest ™ , digested with trypsin, and analyzed by LC/MS/MS as described in “Experimental Procedures”. The normalized relative abundance of a target peptide was calculated as described in “Experimental Procedures”. A , the normalized relative abundance of the BEL-modified S465-containing tryptic peptide (D456-K478) in (R) -BEL- or (S) -BEL-treated samples. B , the normalized relative abundance of the BEL modified C651-containing tryptic peptide (S644-K665) in (R) -BEL- or (S) -BEL-treated samples. C , the normalized relative abundance of the cross-linked peptide in (R) -BEL or (S) -BEL-treated samples. The results indicate that treatment with (S) -BEL results in a higher level of protein modification. By examining the changes in band intensity following SDS-PAGE in the time-course experiment and comparing the levels of modified S465 and C651 as determined by ESI-LC/MS/MS, we found that (S) -BEL is more efficient at covalent modification of iPLA 2 β than (R) -BEL. These observations are in good agreement with the fact that (S) -BEL is a selective inhibitor of iPLA 2 β, and demonstrate the enantioselectivity of (S) - vs. (R)- BEL in the kinetics of covalent modification of iPLA 2 β leading to inhibition of enzymic activity.

Techniques Used: Modification, Purification, Incubation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, SDS Page, Inhibition, Activity Assay

Mass spectral analysis of the tryptic peptides of BEL-modified iPLA 2 β identifying S465 and C651 as sites of modification Purified recombinant iPLA 2 β was incubated with racemic BEL at a molar ratio of 1:1 at 22°C for 3 min. The reaction was terminated by addition of CHCl 3 /CH 3 OH, vortexing, and centrifugation to pellet the precipitated protein. The protein pellet was solubilized in buffer containing RapiGest ™ , digested with trypsin, and analyzed by LC/MS/MS as described in “Experimental Procedures”. A and D, Full mass spectra showing the presence of unique peaks in the BEL-treated sample in the mass range of m/z 878–887 and 905–915, respectively. B, CID mass spectrum (MS 2 ) of the ion at m/z 880.36 (3+) which corresponds to the BEL-modified tryptic peptide, 456 DLFDWVAGTSTGGILALAILHSK 478 . C, CID mass spectrum (MS 3 ) of the ion at m/z 913. E, CID mass spectrum (MS 2 ) of the ion at m/z 908.36 (3+) which corresponds to the BEL-modified tryptic peptide, 644 SPQVPVTCVDVFRPSNPWELAK 665 . F, CID mass spectrum (MS 3 ) of the ion at m/z 1156. MS 2 and MS 3 mass spectra in combination with high mass accuracy (
Figure Legend Snippet: Mass spectral analysis of the tryptic peptides of BEL-modified iPLA 2 β identifying S465 and C651 as sites of modification Purified recombinant iPLA 2 β was incubated with racemic BEL at a molar ratio of 1:1 at 22°C for 3 min. The reaction was terminated by addition of CHCl 3 /CH 3 OH, vortexing, and centrifugation to pellet the precipitated protein. The protein pellet was solubilized in buffer containing RapiGest ™ , digested with trypsin, and analyzed by LC/MS/MS as described in “Experimental Procedures”. A and D, Full mass spectra showing the presence of unique peaks in the BEL-treated sample in the mass range of m/z 878–887 and 905–915, respectively. B, CID mass spectrum (MS 2 ) of the ion at m/z 880.36 (3+) which corresponds to the BEL-modified tryptic peptide, 456 DLFDWVAGTSTGGILALAILHSK 478 . C, CID mass spectrum (MS 3 ) of the ion at m/z 913. E, CID mass spectrum (MS 2 ) of the ion at m/z 908.36 (3+) which corresponds to the BEL-modified tryptic peptide, 644 SPQVPVTCVDVFRPSNPWELAK 665 . F, CID mass spectrum (MS 3 ) of the ion at m/z 1156. MS 2 and MS 3 mass spectra in combination with high mass accuracy (

Techniques Used: Modification, Purification, Recombinant, Incubation, Centrifugation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

9) Product Images from "Improved protease digestion conditions for membrane protein detection"

Article Title: Improved protease digestion conditions for membrane protein detection

Journal: Electrophoresis

doi: 10.1002/elps.201400579

E. coli proteomic study. (4A, 4B) Unique proteins and membrane proteins identified from the membrane protein fraction (MPF) under standard digestion condition, SDS, and MeOH/RapiGest with trypsin and chymotrypsin digestions; in 4B, fractions above the
Figure Legend Snippet: E. coli proteomic study. (4A, 4B) Unique proteins and membrane proteins identified from the membrane protein fraction (MPF) under standard digestion condition, SDS, and MeOH/RapiGest with trypsin and chymotrypsin digestions; in 4B, fractions above the

Techniques Used:

Effects of additives on protease activity. (1A) Effects of MeOH; (1B) Effects of SDS and RapiGest.
Figure Legend Snippet: Effects of additives on protease activity. (1A) Effects of MeOH; (1B) Effects of SDS and RapiGest.

Techniques Used: Activity Assay

Related Articles

Centrifugation:

Article Title: Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach
Article Snippet: Each bait strip was further cut into small pieces and place in a Protein LoBind tube (Eppendorf), washed 3×150 uL of 50 mM ammonium bicarbonate and resuspended in 150 µl of 50 mM ammonium bicarbonate (pH8.0) containing 0.1% Rapigest (Waters, Milford MA). .. Peptides were brought to dryness using vacuum centrifugation and then resuspended in 20 uL 5% acetonitrile, 0.1% formic acid.5 uL of each sample was injected onto a Waters nanoACQUITY UPLC equipped with a 1.7-µm BEH130 C18 reversed-phase column [75 µm inside diameter (ID)×250 mm].

Article Title: Improved protease digestion conditions for membrane protein detection
Article Snippet: Alternatively, MS compatible, acid labile detergents, such as RapiGest (Waters), PPS Silent Surfactant (Protein Discovery), and the noncleavable detergent Invitrosol (Invitrogen) have been developed and are commercially available. .. Upon acidification, RapiGest decomposes into a MS compatible product and a water immiscible product; the latter can be removed by centrifugation.

Expressing:

Article Title: Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65
Article Snippet: Identification of Ser65 phosphorylation of ubiquitin by MS We undertook a SILAC (stable isotope labelling by amino acids in cell culture)-based quantitative phosphoproteomic screen in Flp-In T-Rex HEK-293 cells stably expressing FLAG-empty (L), wild-type (H) or kinase-inactive (M) PINK1–FLAG. .. Mitochondria-containing membrane fractions were enriched by ultracentrifugation and solubilized in 1% RapiGest™ (Waters).

Stable Transfection:

Article Title: Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65
Article Snippet: Identification of Ser65 phosphorylation of ubiquitin by MS We undertook a SILAC (stable isotope labelling by amino acids in cell culture)-based quantitative phosphoproteomic screen in Flp-In T-Rex HEK-293 cells stably expressing FLAG-empty (L), wild-type (H) or kinase-inactive (M) PINK1–FLAG. .. Mitochondria-containing membrane fractions were enriched by ultracentrifugation and solubilized in 1% RapiGest™ (Waters).

Bradford Assay:

Article Title: mTORC1 promotes T-bet phosphorylation to regulate Th1 differentiation
Article Snippet: Frozen pellets were thawed on ice and lysed with 0.15% Rapigest (Waters) in 100 mM NH4 HCO3 (pH 8.4) supplemented with phosphatase inhibitors 10 mM Na4 O7 P2 , 50 mM NaF and 1 mM Na3 VO4. .. Protein concentration of resulting lysates was measured by Bradford assay and proteins were digested overnight with sequencing grade modified trypsin (Promega) at 1:50 trypsin:protein ratio.

Incubation:

Article Title: Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach
Article Snippet: Each bait strip was further cut into small pieces and place in a Protein LoBind tube (Eppendorf), washed 3×150 uL of 50 mM ammonium bicarbonate and resuspended in 150 µl of 50 mM ammonium bicarbonate (pH8.0) containing 0.1% Rapigest (Waters, Milford MA). .. Samples were reduced in 10 mM dithiolthreitol at 40 C for 20 min and alkylated with iodoacetamide at 20 mM at room temperature for 40 min. On-membrane digestion was performed by adding 500 ng trypsin (Promega) and incubation at 37 C for 18 hr.

Article Title: Influence of a Hyperglycemic Microenvironment on a Diabetic Versus Healthy Rat Vascular Endothelium Reveals Distinguishable Mechanistic and Phenotypic Responses
Article Snippet: The lysate was then diluted in 6 mL membrane preparation buffer (280 mmol/L sucrose, 50 mmol/L MES (pH 6.5), 450 mmol/L NaCl, and 10 mmol/L MgCl2 ) and incubated on ice for 10 min. .. Supernatant from the membrane wash was removed and membrane fractions were resuspended in 50 mM ammonium bicarbonate, pH 8.0, 0.1% RapiGest (Waters), and 10 mM dithiolthreitol (DTT) for 60 min at 37°C in a thermomixer at 500 rpm; cytosolic fractions were also reduced in this manner.

Article Title: Site-Dependent Degradation of a Non-Cleavable Auristatin-Based Linker-Payload in Rodent Plasma and Its Effect on ADC Efficacy
Article Snippet: LC/MS/MS analysis of ADC tryptic digestions ADCs (100 μg) were solubilized in 0.2% RapiGest (Waters Corp, 186001861) in 20 mM ammonium bicarbonate. .. The samples were then incubated at 80°C for 15 min. Dithiothreitol (DTT, 20 mM final concentration) was added and the samples were incubated at 60°C for 30 min to reduce disulfide bonds.

Article Title: Quantification of Fucosylated Hemopexin and Complement Factor H in Plasma of Patients with Liver Disease
Article Snippet: Briefly, 200 μL of plasma was diluted 1:2 with PBS, loaded to 200 μL of hemin–agarose suspension (Sigma-Aldrich, St. Louis, MO), and incubated overnight at 4 °C. .. Purified proteins were dried in a CentriVap vacuum concentrator (Labconco, Kansas City, MO), reconstituted in 50 μL of 50 mM NH4 HCO3 , pH 8.0, with 0.05% RapiGest (Waters, Milford, MA), and stored at −20 °C until use.

Article Title: Affinity pulldown of γ-secretase and associated proteins from human and rat brain
Article Snippet: The samples were incubated with SA-beads which were pre-equilibrated by buffer A with 0.5% CHAPSO for 2 hr at 4°C. .. The captured γ-secretase complex was eluted from the resin by buffer A containing 100 mM DTT and 0.5% CHAPSO, SDS sample buffer, or 0.01% RapiGest (Waters, Milford, MA, USA) in 10 mM ammonium bicarbonate supplemented with 10 mM DTT.

Article Title: mTORC1 promotes T-bet phosphorylation to regulate Th1 differentiation
Article Snippet: Frozen pellets were thawed on ice and lysed with 0.15% Rapigest (Waters) in 100 mM NH4 HCO3 (pH 8.4) supplemented with phosphatase inhibitors 10 mM Na4 O7 P2 , 50 mM NaF and 1 mM Na3 VO4. .. Digested samples were acidified to pH of 2.5–3 by 10% trifluoroacetic acid then incubated at 37°C for 1 hour.

Article Title: Proteomic Analysis of the Extracellular Matrix Produced by Mesenchymal Stromal Cells: Implications for Cell Therapy Mechanism
Article Snippet: In brief, 500 µg of precipitated SDS/urea-soluble and an unknown amount of SDS/urea-insoluble SB623- and MSC-derived ECM from the same human donor (one SDS/urea-soluble fraction for SB623, ; two fractions for SDS/urea-soluble and two fractions of SDS/urea-insoluble from each MSC- and SB623-derived ECM preparations, ) were resuspended in 50 µl of 0.1% Rapigest (Waters) and vortexed. .. The reduced ECM samples were cooled to room temperature and alkylated with iodoacetamideto final concentration of 15 mM, and then incubated in the dark for 30 min at room temperature.

Article Title: Mechanism-based Inhibition of iPLA2β Demonstrates a Highly Reactive Cysteine Residue (C651) That Interacts with the Active Site: Mass Spectrometric Elucidation of the Mechanisms of Underlying Inhibition
Article Snippet: Purified iPLA2 β (5 μM) was incubated in the presence or absence of oleoyl-CoA (50 μM) for 60 min at 30°C. .. Protein was precipitated with CHCl3 /CH3 OH as previously described , dried using a Speed-Vac, and solubilized in 25 mM NH4 HCO3 containing 0.1% RapiGest™ (Waters, Milford, MA).

Stripping Membranes:

Article Title: Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach
Article Snippet: .. Each bait strip was further cut into small pieces and place in a Protein LoBind tube (Eppendorf), washed 3×150 uL of 50 mM ammonium bicarbonate and resuspended in 150 µl of 50 mM ammonium bicarbonate (pH8.0) containing 0.1% Rapigest (Waters, Milford MA). .. Samples were reduced in 10 mM dithiolthreitol at 40 C for 20 min and alkylated with iodoacetamide at 20 mM at room temperature for 40 min. On-membrane digestion was performed by adding 500 ng trypsin (Promega) and incubation at 37 C for 18 hr.

Activity Assay:

Article Title: Improved protease digestion conditions for membrane protein detection
Article Snippet: Nonetheless, SDS negatively affects protease activity, interferes with LC separation and has poor MS compatibility. .. Alternatively, MS compatible, acid labile detergents, such as RapiGest (Waters), PPS Silent Surfactant (Protein Discovery), and the noncleavable detergent Invitrosol (Invitrogen) have been developed and are commercially available.

Article Title: Improved protease digestion conditions for membrane protein detection
Article Snippet: Paragraph title: 2.2 Protease activity determination ... Protease activities in the presence of different concentrations of MeOH (Mallinckrodt Chemicals, Phillipsburg, NJ), RapiGest (Waters, Milford, MA) and SDS (Bio-Rad Laboratories, Hercules, CA) were normalized to protease activities without any additives.

Mass Spectrometry:

Article Title: Influence of a Hyperglycemic Microenvironment on a Diabetic Versus Healthy Rat Vascular Endothelium Reveals Distinguishable Mechanistic and Phenotypic Responses
Article Snippet: Paragraph title: Preparation of Samples for Mass Spectrometry Analysis ... Supernatant from the membrane wash was removed and membrane fractions were resuspended in 50 mM ammonium bicarbonate, pH 8.0, 0.1% RapiGest (Waters), and 10 mM dithiolthreitol (DTT) for 60 min at 37°C in a thermomixer at 500 rpm; cytosolic fractions were also reduced in this manner.

Article Title: Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis
Article Snippet: The remaining chemicals and reagents were purchased from individual supplier: sequencing grade modified trypsin (Promega, Madison, WI), formaldehyde (D2, 98%, CD2 O, Cambridge Isotope Laboratories, Andover, MA), ammonium hydroxide (NH4 OH, BAKER ANALYZED, Deventer, the Netherlands), PMSF (AppliChem, Darmstadt, Germany), 2-Amino-2-hydroxymethyl-propane-1,3-diol (Tris, VWR International, Leuven, Belgium), Rapigest (Waters, Milford, MA), titanium dioxide beads (TiO2 , GL Sciences Inc., Tokyo, Japan). .. Solutions for mass spectrometry analysis were prepared using LiChrosolv water (Merck KGaA, Darmstadt, Germany).

Article Title: 5-azacytidine inhibits nonsense-mediated decay in a MYC-dependent fashion
Article Snippet: Paragraph title: Dimethyl labeling and quantitative mass spectrometry ... After lysis of cells in 0.1% RapiGest (Waters) and 50 mM (NH4 ) HCO3 , extracted proteins were subsequently reduced and alkylated with 5 mM DTT and 10 mM iodoacetamide and digested overnight with sequencing-grade modified trypsin (Promega).

Article Title: Improved protease digestion conditions for membrane protein detection
Article Snippet: .. Alternatively, MS compatible, acid labile detergents, such as RapiGest (Waters), PPS Silent Surfactant (Protein Discovery), and the noncleavable detergent Invitrosol (Invitrogen) have been developed and are commercially available. ..

Article Title: Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65
Article Snippet: Paragraph title: Identification of Ser65 phosphorylation of ubiquitin by MS ... Mitochondria-containing membrane fractions were enriched by ultracentrifugation and solubilized in 1% RapiGest™ (Waters).

Article Title: mTORC1 promotes T-bet phosphorylation to regulate Th1 differentiation
Article Snippet: Paragraph title: Sample preparation for mass spectrometry analysis ... Frozen pellets were thawed on ice and lysed with 0.15% Rapigest (Waters) in 100 mM NH4 HCO3 (pH 8.4) supplemented with phosphatase inhibitors 10 mM Na4 O7 P2 , 50 mM NaF and 1 mM Na3 VO4.

Article Title: Proteomic Analysis of the Extracellular Matrix Produced by Mesenchymal Stromal Cells: Implications for Cell Therapy Mechanism
Article Snippet: Surfactant Assisted In-Solution Digests (SAISD) SAISD was used for the preparation of SDS/urea-soluble and insoluble SB623-and MSC-derived ECM for nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS). .. In brief, 500 µg of precipitated SDS/urea-soluble and an unknown amount of SDS/urea-insoluble SB623- and MSC-derived ECM from the same human donor (one SDS/urea-soluble fraction for SB623, ; two fractions for SDS/urea-soluble and two fractions of SDS/urea-insoluble from each MSC- and SB623-derived ECM preparations, ) were resuspended in 50 µl of 0.1% Rapigest (Waters) and vortexed.

BIA-KA:

Article Title: Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis
Article Snippet: Pierce bicinchoninic acid protein concentration assay (BCA), albumin standard, Pierce protease and phosphatase inhibitor, acetonitrile (ACN, LC-MS grade) were purchased from Thermo Fisher Scientific (Rockford, IL). .. The remaining chemicals and reagents were purchased from individual supplier: sequencing grade modified trypsin (Promega, Madison, WI), formaldehyde (D2, 98%, CD2 O, Cambridge Isotope Laboratories, Andover, MA), ammonium hydroxide (NH4 OH, BAKER ANALYZED, Deventer, the Netherlands), PMSF (AppliChem, Darmstadt, Germany), 2-Amino-2-hydroxymethyl-propane-1,3-diol (Tris, VWR International, Leuven, Belgium), Rapigest (Waters, Milford, MA), titanium dioxide beads (TiO2 , GL Sciences Inc., Tokyo, Japan).

Modification:

Article Title: Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach
Article Snippet: Each bait strip was further cut into small pieces and place in a Protein LoBind tube (Eppendorf), washed 3×150 uL of 50 mM ammonium bicarbonate and resuspended in 150 µl of 50 mM ammonium bicarbonate (pH8.0) containing 0.1% Rapigest (Waters, Milford MA). .. Polymer contamination, mostly polyethylene glycol, were removed using ProteoSpin detergent removal spin-columns (Norgen Biotek, ON Canada, product 10200) with a modified elution buffer solution of 5% aqueous ammonia in 50 mM ammonium bicarbonate, pH 10.0).

Article Title: Site-Dependent Degradation of a Non-Cleavable Auristatin-Based Linker-Payload in Rodent Plasma and Its Effect on ADC Efficacy
Article Snippet: LC/MS/MS analysis of ADC tryptic digestions ADCs (100 μg) were solubilized in 0.2% RapiGest (Waters Corp, 186001861) in 20 mM ammonium bicarbonate. .. Modified trypsin (Promega, cat#V5111) was added (1:100 enzyme/substrate) and the samples were incubated at 37°C overnight.

Article Title: Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis
Article Snippet: .. The remaining chemicals and reagents were purchased from individual supplier: sequencing grade modified trypsin (Promega, Madison, WI), formaldehyde (D2, 98%, CD2 O, Cambridge Isotope Laboratories, Andover, MA), ammonium hydroxide (NH4 OH, BAKER ANALYZED, Deventer, the Netherlands), PMSF (AppliChem, Darmstadt, Germany), 2-Amino-2-hydroxymethyl-propane-1,3-diol (Tris, VWR International, Leuven, Belgium), Rapigest (Waters, Milford, MA), titanium dioxide beads (TiO2 , GL Sciences Inc., Tokyo, Japan). .. Sucrose buffer: 320 mM sucrose, 5 mM Hepes, pH=7.4; sodium buffer: 10 mM glucose, 5 mM KCl, 140 mM NaCl, 5 mM NaHCO3 , 1 mM MgCl2 , 1.2 mM Na2 HPO4 , 20 mM Hepes, pH=7.4; lysis buffer: 50 mM Tris, 150 mM NaCl, 1% Nonidet P40, pH=7.4 containing Pierce protease and phosphatase inhibitors; 6%, 9% and 13% Ficoll solutions were prepared in sucrose buffer (pH=7.4).

Article Title: 5-azacytidine inhibits nonsense-mediated decay in a MYC-dependent fashion
Article Snippet: .. After lysis of cells in 0.1% RapiGest (Waters) and 50 mM (NH4 ) HCO3 , extracted proteins were subsequently reduced and alkylated with 5 mM DTT and 10 mM iodoacetamide and digested overnight with sequencing-grade modified trypsin (Promega). .. Peptides were labeled on SepPak C18 cartridges (Waters) with labeling reagent (light and intermediate, with CH2 O (Fisher) plus NaBH3 CN (Fluka) or CD2 O (Isotec) plus NaBH3 CN, respectively) as described in Boersemaet al ( ).

Article Title: mTORC1 promotes T-bet phosphorylation to regulate Th1 differentiation
Article Snippet: Frozen pellets were thawed on ice and lysed with 0.15% Rapigest (Waters) in 100 mM NH4 HCO3 (pH 8.4) supplemented with phosphatase inhibitors 10 mM Na4 O7 P2 , 50 mM NaF and 1 mM Na3 VO4. .. Protein concentration of resulting lysates was measured by Bradford assay and proteins were digested overnight with sequencing grade modified trypsin (Promega) at 1:50 trypsin:protein ratio.

Flow Cytometry:

Article Title: Quantification of Fucosylated Hemopexin and Complement Factor H in Plasma of Patients with Liver Disease
Article Snippet: Bound glycoproteins were eluted with 0.2 M citric acid, pH 2.0, neutralized with 1 M Tris-HCl, pH 9.5, precipitated with methanol/chloroform as described, solubilized in solvent A (2% acetonitrile (ACN), 0.1% TFA), and separated on an mRP Hi-Recovery Protein 4.6 × 50 mm C18 column (Agilent Technologies, Santa Clara, CA) heated to 40 °C at a flow rate of 0.75 mL/min as follows: 0–5 min 1% B, 10 min 35% B, 25 min 45% B, 30 min 100% B, 31 min 100% B, 33 min 1% B, 45 min 1% B (B: 98% ACN, 0.08% TFA). .. Purified proteins were dried in a CentriVap vacuum concentrator (Labconco, Kansas City, MO), reconstituted in 50 μL of 50 mM NH4 HCO3 , pH 8.0, with 0.05% RapiGest (Waters, Milford, MA), and stored at −20 °C until use.

Chromatography:

Article Title: Proteomic Analysis of the Extracellular Matrix Produced by Mesenchymal Stromal Cells: Implications for Cell Therapy Mechanism
Article Snippet: Surfactant Assisted In-Solution Digests (SAISD) SAISD was used for the preparation of SDS/urea-soluble and insoluble SB623-and MSC-derived ECM for nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS). .. In brief, 500 µg of precipitated SDS/urea-soluble and an unknown amount of SDS/urea-insoluble SB623- and MSC-derived ECM from the same human donor (one SDS/urea-soluble fraction for SB623, ; two fractions for SDS/urea-soluble and two fractions of SDS/urea-insoluble from each MSC- and SB623-derived ECM preparations, ) were resuspended in 50 µl of 0.1% Rapigest (Waters) and vortexed.

Solubility:

Article Title: Improved protease digestion conditions for membrane protein detection
Article Snippet: Different strategies have been developed to improve membrane protein solubility, including the addition of detergents or organic solvents [ , , ]. .. Alternatively, MS compatible, acid labile detergents, such as RapiGest (Waters), PPS Silent Surfactant (Protein Discovery), and the noncleavable detergent Invitrosol (Invitrogen) have been developed and are commercially available.

Peptide Microarray:

Article Title: Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach
Article Snippet: Mass Spectrometric Analysis Following the PAX procedure, the peptide array membrane was cut and separated into bait strips. .. Each bait strip was further cut into small pieces and place in a Protein LoBind tube (Eppendorf), washed 3×150 uL of 50 mM ammonium bicarbonate and resuspended in 150 µl of 50 mM ammonium bicarbonate (pH8.0) containing 0.1% Rapigest (Waters, Milford MA).

Protein Concentration:

Article Title: Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis
Article Snippet: Pierce bicinchoninic acid protein concentration assay (BCA), albumin standard, Pierce protease and phosphatase inhibitor, acetonitrile (ACN, LC-MS grade) were purchased from Thermo Fisher Scientific (Rockford, IL). .. The remaining chemicals and reagents were purchased from individual supplier: sequencing grade modified trypsin (Promega, Madison, WI), formaldehyde (D2, 98%, CD2 O, Cambridge Isotope Laboratories, Andover, MA), ammonium hydroxide (NH4 OH, BAKER ANALYZED, Deventer, the Netherlands), PMSF (AppliChem, Darmstadt, Germany), 2-Amino-2-hydroxymethyl-propane-1,3-diol (Tris, VWR International, Leuven, Belgium), Rapigest (Waters, Milford, MA), titanium dioxide beads (TiO2 , GL Sciences Inc., Tokyo, Japan).

Article Title: mTORC1 promotes T-bet phosphorylation to regulate Th1 differentiation
Article Snippet: Frozen pellets were thawed on ice and lysed with 0.15% Rapigest (Waters) in 100 mM NH4 HCO3 (pH 8.4) supplemented with phosphatase inhibitors 10 mM Na4 O7 P2 , 50 mM NaF and 1 mM Na3 VO4. .. Protein concentration of resulting lysates was measured by Bradford assay and proteins were digested overnight with sequencing grade modified trypsin (Promega) at 1:50 trypsin:protein ratio.

Sequencing:

Article Title: Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis
Article Snippet: .. The remaining chemicals and reagents were purchased from individual supplier: sequencing grade modified trypsin (Promega, Madison, WI), formaldehyde (D2, 98%, CD2 O, Cambridge Isotope Laboratories, Andover, MA), ammonium hydroxide (NH4 OH, BAKER ANALYZED, Deventer, the Netherlands), PMSF (AppliChem, Darmstadt, Germany), 2-Amino-2-hydroxymethyl-propane-1,3-diol (Tris, VWR International, Leuven, Belgium), Rapigest (Waters, Milford, MA), titanium dioxide beads (TiO2 , GL Sciences Inc., Tokyo, Japan). .. Sucrose buffer: 320 mM sucrose, 5 mM Hepes, pH=7.4; sodium buffer: 10 mM glucose, 5 mM KCl, 140 mM NaCl, 5 mM NaHCO3 , 1 mM MgCl2 , 1.2 mM Na2 HPO4 , 20 mM Hepes, pH=7.4; lysis buffer: 50 mM Tris, 150 mM NaCl, 1% Nonidet P40, pH=7.4 containing Pierce protease and phosphatase inhibitors; 6%, 9% and 13% Ficoll solutions were prepared in sucrose buffer (pH=7.4).

Article Title: 5-azacytidine inhibits nonsense-mediated decay in a MYC-dependent fashion
Article Snippet: .. After lysis of cells in 0.1% RapiGest (Waters) and 50 mM (NH4 ) HCO3 , extracted proteins were subsequently reduced and alkylated with 5 mM DTT and 10 mM iodoacetamide and digested overnight with sequencing-grade modified trypsin (Promega). .. Peptides were labeled on SepPak C18 cartridges (Waters) with labeling reagent (light and intermediate, with CH2 O (Fisher) plus NaBH3 CN (Fluka) or CD2 O (Isotec) plus NaBH3 CN, respectively) as described in Boersemaet al ( ).

Article Title: mTORC1 promotes T-bet phosphorylation to regulate Th1 differentiation
Article Snippet: Frozen pellets were thawed on ice and lysed with 0.15% Rapigest (Waters) in 100 mM NH4 HCO3 (pH 8.4) supplemented with phosphatase inhibitors 10 mM Na4 O7 P2 , 50 mM NaF and 1 mM Na3 VO4. .. Protein concentration of resulting lysates was measured by Bradford assay and proteins were digested overnight with sequencing grade modified trypsin (Promega) at 1:50 trypsin:protein ratio.

Sonication:

Article Title: Influence of a Hyperglycemic Microenvironment on a Diabetic Versus Healthy Rat Vascular Endothelium Reveals Distinguishable Mechanistic and Phenotypic Responses
Article Snippet: Samples were diluted to 4 mL in hypotonic lysis buffer and mechanically lysed by twenty passages, followed by a 30 min water bath sonication. .. Supernatant from the membrane wash was removed and membrane fractions were resuspended in 50 mM ammonium bicarbonate, pH 8.0, 0.1% RapiGest (Waters), and 10 mM dithiolthreitol (DTT) for 60 min at 37°C in a thermomixer at 500 rpm; cytosolic fractions were also reduced in this manner.

Article Title: Rapid, Optimized Interactomic Screening
Article Snippet: .. The precipitate was resuspended in 50 mM ammonium bicarbonate, 0.1% w/v RapiGest (Waters) via bath sonication with heating (20 min at 70°C, followed by 2min at 95°C). .. The proteins were digested with trypsin (Promega) overnight.

Article Title: Mechanism-based Inhibition of iPLA2β Demonstrates a Highly Reactive Cysteine Residue (C651) That Interacts with the Active Site: Mass Spectrometric Elucidation of the Mechanisms of Underlying Inhibition
Article Snippet: Protein was precipitated with CHCl3 /CH3 OH as previously described , dried using a Speed-Vac, and solubilized in 25 mM NH4 HCO3 containing 0.1% RapiGest™ (Waters, Milford, MA). .. The supernatant was removed and the pellet was resuspended in 70% isopropanol by a combination of vortexing and sonication.

Injection:

Article Title: Peptide Array X-Linking (PAX): A New Peptide-Protein Identification Approach
Article Snippet: Each bait strip was further cut into small pieces and place in a Protein LoBind tube (Eppendorf), washed 3×150 uL of 50 mM ammonium bicarbonate and resuspended in 150 µl of 50 mM ammonium bicarbonate (pH8.0) containing 0.1% Rapigest (Waters, Milford MA). .. Peptides were brought to dryness using vacuum centrifugation and then resuspended in 20 uL 5% acetonitrile, 0.1% formic acid.5 uL of each sample was injected onto a Waters nanoACQUITY UPLC equipped with a 1.7-µm BEH130 C18 reversed-phase column [75 µm inside diameter (ID)×250 mm].

Cellular Antioxidant Activity Assay:

Article Title: Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis
Article Snippet: Calcium chloride (CaCl2 ), nicotinamide adenine dinucleotide phosphate hydrate (NADPx1H2 0), ammonium hydrogen carbonate (NH4 HCO3 ), 2-chloroacetamide (CAA), acetone (Chromasolv grade), glutamate dehydrogenase (GluDH), formic acid (FA), dithiothreitol (DTT), pepstatin-A, trifluoroacetic acid (TFA), Nonidet P40, tetraethylammonium bromide (TEAB), 2,5-dihydroxybenzoic acid (DHB), ammonium formate (NH4 HCO2 ), Ficoll PM 400, sodium cyanoborohydride (NaBD3 CN), formaldehyde (CH2 O) were purchased from Sigma-Aldrich (Steinheim, Germany). .. The remaining chemicals and reagents were purchased from individual supplier: sequencing grade modified trypsin (Promega, Madison, WI), formaldehyde (D2, 98%, CD2 O, Cambridge Isotope Laboratories, Andover, MA), ammonium hydroxide (NH4 OH, BAKER ANALYZED, Deventer, the Netherlands), PMSF (AppliChem, Darmstadt, Germany), 2-Amino-2-hydroxymethyl-propane-1,3-diol (Tris, VWR International, Leuven, Belgium), Rapigest (Waters, Milford, MA), titanium dioxide beads (TiO2 , GL Sciences Inc., Tokyo, Japan).

Fluorescence:

Article Title: Improved protease digestion conditions for membrane protein detection
Article Snippet: Protease activities in the presence of different concentrations of MeOH (Mallinckrodt Chemicals, Phillipsburg, NJ), RapiGest (Waters, Milford, MA) and SDS (Bio-Rad Laboratories, Hercules, CA) were normalized to protease activities without any additives. .. Fluorescence was measured on a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA).

Magnetic Beads:

Article Title: Affinity pulldown of γ-secretase and associated proteins from human and rat brain
Article Snippet: The samples were incubated with SA-conjugated sepharose beads (GE Healthcare) or SA-conjugated magnetic beads (Invitrogen) to remove endogenous biotinylated proteins for 16 hr at 4°C. .. The captured γ-secretase complex was eluted from the resin by buffer A containing 100 mM DTT and 0.5% CHAPSO, SDS sample buffer, or 0.01% RapiGest (Waters, Milford, MA, USA) in 10 mM ammonium bicarbonate supplemented with 10 mM DTT.

Isolation:

Article Title: Quantification of Fucosylated Hemopexin and Complement Factor H in Plasma of Patients with Liver Disease
Article Snippet: Paragraph title: Isolation of Glycoproteins from Plasma ... Purified proteins were dried in a CentriVap vacuum concentrator (Labconco, Kansas City, MO), reconstituted in 50 μL of 50 mM NH4 HCO3 , pH 8.0, with 0.05% RapiGest (Waters, Milford, MA), and stored at −20 °C until use.

Tandem Mass Spectroscopy:

Article Title: Improved protease digestion conditions for membrane protein detection
Article Snippet: Thus, SDS is typically removed or diluted prior to digestion and MS/MS data acquisition; however, SDS has high affinity for proteins and peptides and is very difficult to remove. .. Alternatively, MS compatible, acid labile detergents, such as RapiGest (Waters), PPS Silent Surfactant (Protein Discovery), and the noncleavable detergent Invitrosol (Invitrogen) have been developed and are commercially available.

Labeling:

Article Title: 5-azacytidine inhibits nonsense-mediated decay in a MYC-dependent fashion
Article Snippet: Paragraph title: Dimethyl labeling and quantitative mass spectrometry ... After lysis of cells in 0.1% RapiGest (Waters) and 50 mM (NH4 ) HCO3 , extracted proteins were subsequently reduced and alkylated with 5 mM DTT and 10 mM iodoacetamide and digested overnight with sequencing-grade modified trypsin (Promega).

Purification:

Article Title: Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis
Article Snippet: The remaining chemicals and reagents were purchased from individual supplier: sequencing grade modified trypsin (Promega, Madison, WI), formaldehyde (D2, 98%, CD2 O, Cambridge Isotope Laboratories, Andover, MA), ammonium hydroxide (NH4 OH, BAKER ANALYZED, Deventer, the Netherlands), PMSF (AppliChem, Darmstadt, Germany), 2-Amino-2-hydroxymethyl-propane-1,3-diol (Tris, VWR International, Leuven, Belgium), Rapigest (Waters, Milford, MA), titanium dioxide beads (TiO2 , GL Sciences Inc., Tokyo, Japan). .. The buffers were prepared using nanopure water obtained with an electric resistance of greater that 18 MΩ from a Mili-Q purification system (MerckMillipore, Darmstadt, Germany).

Article Title: Quantification of Fucosylated Hemopexin and Complement Factor H in Plasma of Patients with Liver Disease
Article Snippet: .. Purified proteins were dried in a CentriVap vacuum concentrator (Labconco, Kansas City, MO), reconstituted in 50 μL of 50 mM NH4 HCO3 , pH 8.0, with 0.05% RapiGest (Waters, Milford, MA), and stored at −20 °C until use. ..

Article Title: Mechanism-based Inhibition of iPLA2β Demonstrates a Highly Reactive Cysteine Residue (C651) That Interacts with the Active Site: Mass Spectrometric Elucidation of the Mechanisms of Underlying Inhibition
Article Snippet: Purified iPLA2 β (5 μM) was incubated in the presence or absence of oleoyl-CoA (50 μM) for 60 min at 30°C. .. Protein was precipitated with CHCl3 /CH3 OH as previously described , dried using a Speed-Vac, and solubilized in 25 mM NH4 HCO3 containing 0.1% RapiGest™ (Waters, Milford, MA).

Protein Extraction:

Article Title: The beauty of being (label)-free: sample preparation methods for SWATH-MS and next-generation targeted proteomics
Article Snippet: .. Protocol 5: RapiGest (Waters (UK), RapiGest-including version of a protocol based on der Haar, T.Optimized protein extraction for quantitative proteomics of yeasts . ..

Chloramphenicol Acetyltransferase Assay:

Article Title: Site-Dependent Degradation of a Non-Cleavable Auristatin-Based Linker-Payload in Rodent Plasma and Its Effect on ADC Efficacy
Article Snippet: LC/MS/MS analysis of ADC tryptic digestions ADCs (100 μg) were solubilized in 0.2% RapiGest (Waters Corp, 186001861) in 20 mM ammonium bicarbonate. .. Modified trypsin (Promega, catV5111) was added (1:100 enzyme/substrate) and the samples were incubated at 37°C overnight.

Software:

Article Title: Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65
Article Snippet: Mitochondria-containing membrane fractions were enriched by ultracentrifugation and solubilized in 1% RapiGest™ (Waters). .. Data were analysed using Maxquant 1.3.0.5 [ ] and Xcalibur software (Thermo Fisher).

Affinity Chromatography:

Article Title: Quantification of Fucosylated Hemopexin and Complement Factor H in Plasma of Patients with Liver Disease
Article Snippet: HPX and CFH were isolated from plasma by hemin affinity chromatography as described previously with slight modifications. .. Purified proteins were dried in a CentriVap vacuum concentrator (Labconco, Kansas City, MO), reconstituted in 50 μL of 50 mM NH4 HCO3 , pH 8.0, with 0.05% RapiGest (Waters, Milford, MA), and stored at −20 °C until use.

Sample Prep:

Article Title: mTORC1 promotes T-bet phosphorylation to regulate Th1 differentiation
Article Snippet: Paragraph title: Sample preparation for mass spectrometry analysis ... Frozen pellets were thawed on ice and lysed with 0.15% Rapigest (Waters) in 100 mM NH4 HCO3 (pH 8.4) supplemented with phosphatase inhibitors 10 mM Na4 O7 P2 , 50 mM NaF and 1 mM Na3 VO4.

Concentration Assay:

Article Title: Site-Dependent Degradation of a Non-Cleavable Auristatin-Based Linker-Payload in Rodent Plasma and Its Effect on ADC Efficacy
Article Snippet: LC/MS/MS analysis of ADC tryptic digestions ADCs (100 μg) were solubilized in 0.2% RapiGest (Waters Corp, 186001861) in 20 mM ammonium bicarbonate. .. The samples were then incubated at 80°C for 15 min. Dithiothreitol (DTT, 20 mM final concentration) was added and the samples were incubated at 60°C for 30 min to reduce disulfide bonds.

Article Title: Improved protease digestion conditions for membrane protein detection
Article Snippet: The concentration of SDS used in shotgun proteomics varies from 0.1% to up to 1%. .. Alternatively, MS compatible, acid labile detergents, such as RapiGest (Waters), PPS Silent Surfactant (Protein Discovery), and the noncleavable detergent Invitrosol (Invitrogen) have been developed and are commercially available.

Article Title: Proteomic Analysis of the Extracellular Matrix Produced by Mesenchymal Stromal Cells: Implications for Cell Therapy Mechanism
Article Snippet: In brief, 500 µg of precipitated SDS/urea-soluble and an unknown amount of SDS/urea-insoluble SB623- and MSC-derived ECM from the same human donor (one SDS/urea-soluble fraction for SB623, ; two fractions for SDS/urea-soluble and two fractions of SDS/urea-insoluble from each MSC- and SB623-derived ECM preparations, ) were resuspended in 50 µl of 0.1% Rapigest (Waters) and vortexed. .. DTT was added to the ECM samples to a final concentration of 5 mM and heated at 60°C for 30 min.

Article Title: Mechanism-based Inhibition of iPLA2β Demonstrates a Highly Reactive Cysteine Residue (C651) That Interacts with the Active Site: Mass Spectrometric Elucidation of the Mechanisms of Underlying Inhibition
Article Snippet: Protein was precipitated with CHCl3 /CH3 OH as previously described , dried using a Speed-Vac, and solubilized in 25 mM NH4 HCO3 containing 0.1% RapiGest™ (Waters, Milford, MA). .. After termination of the trypsinolysis by addition of trifluoroacetic acid (0.5% final concentration), the samples were further incubated at 37°C for 45 min and centrifuged at 13,000 rpm for 15 min.

Liquid Chromatography with Mass Spectroscopy:

Article Title: Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis
Article Snippet: Pierce bicinchoninic acid protein concentration assay (BCA), albumin standard, Pierce protease and phosphatase inhibitor, acetonitrile (ACN, LC-MS grade) were purchased from Thermo Fisher Scientific (Rockford, IL). .. The remaining chemicals and reagents were purchased from individual supplier: sequencing grade modified trypsin (Promega, Madison, WI), formaldehyde (D2, 98%, CD2 O, Cambridge Isotope Laboratories, Andover, MA), ammonium hydroxide (NH4 OH, BAKER ANALYZED, Deventer, the Netherlands), PMSF (AppliChem, Darmstadt, Germany), 2-Amino-2-hydroxymethyl-propane-1,3-diol (Tris, VWR International, Leuven, Belgium), Rapigest (Waters, Milford, MA), titanium dioxide beads (TiO2 , GL Sciences Inc., Tokyo, Japan).

Article Title: Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65
Article Snippet: Mitochondria-containing membrane fractions were enriched by ultracentrifugation and solubilized in 1% RapiGest™ (Waters). .. Digested peptides were subjected to TiO2 phosphopeptide enrichment [ , ] and analysed by LC–MS on an Orbitrap Velos Pro (Thermo Fisher).

Lysis:

Article Title: Influence of a Hyperglycemic Microenvironment on a Diabetic Versus Healthy Rat Vascular Endothelium Reveals Distinguishable Mechanistic and Phenotypic Responses
Article Snippet: Samples were diluted to 4 mL in hypotonic lysis buffer and mechanically lysed by twenty passages, followed by a 30 min water bath sonication. .. Supernatant from the membrane wash was removed and membrane fractions were resuspended in 50 mM ammonium bicarbonate, pH 8.0, 0.1% RapiGest (Waters), and 10 mM dithiolthreitol (DTT) for 60 min at 37°C in a thermomixer at 500 rpm; cytosolic fractions were also reduced in this manner.

Article Title: Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis
Article Snippet: The remaining chemicals and reagents were purchased from individual supplier: sequencing grade modified trypsin (Promega, Madison, WI), formaldehyde (D2, 98%, CD2 O, Cambridge Isotope Laboratories, Andover, MA), ammonium hydroxide (NH4 OH, BAKER ANALYZED, Deventer, the Netherlands), PMSF (AppliChem, Darmstadt, Germany), 2-Amino-2-hydroxymethyl-propane-1,3-diol (Tris, VWR International, Leuven, Belgium), Rapigest (Waters, Milford, MA), titanium dioxide beads (TiO2 , GL Sciences Inc., Tokyo, Japan). .. Sucrose buffer: 320 mM sucrose, 5 mM Hepes, pH=7.4; sodium buffer: 10 mM glucose, 5 mM KCl, 140 mM NaCl, 5 mM NaHCO3 , 1 mM MgCl2 , 1.2 mM Na2 HPO4 , 20 mM Hepes, pH=7.4; lysis buffer: 50 mM Tris, 150 mM NaCl, 1% Nonidet P40, pH=7.4 containing Pierce protease and phosphatase inhibitors; 6%, 9% and 13% Ficoll solutions were prepared in sucrose buffer (pH=7.4).

Article Title: 5-azacytidine inhibits nonsense-mediated decay in a MYC-dependent fashion
Article Snippet: .. After lysis of cells in 0.1% RapiGest (Waters) and 50 mM (NH4 ) HCO3 , extracted proteins were subsequently reduced and alkylated with 5 mM DTT and 10 mM iodoacetamide and digested overnight with sequencing-grade modified trypsin (Promega). .. Peptides were labeled on SepPak C18 cartridges (Waters) with labeling reagent (light and intermediate, with CH2 O (Fisher) plus NaBH3 CN (Fluka) or CD2 O (Isotec) plus NaBH3 CN, respectively) as described in Boersemaet al ( ).

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    Waters Corporation reagent rapigest sf
    In solution digestion leads to stable results in label-free proteomics. ( a ) The distribution maximum of coefficients of variation (CV) of the selected protocols varies between 0.075 and 0.2. CV values obtained for protocol triplicates are shown as two-dimensional distribution histograms (‘violin plots’). Quantification in DDA experiments was consistent over the dynamic range, as CV values only marginally changed when filtering by peptides according to their abundance (80%, 60%, 40% or 20%). CV likelihood maxima of all protocols were below 20%, while RapidACN led to the most reproducible results (CV = 7%) (n = 3). ( b ) Stability in a quantification experiment is improved by data-independent acquisition. CV values for the same set of peptides measured with SWATH and DDA using the <t>RapiGest</t> protocol, as shown in a two-dimensional distribution histogram. Whereas there was a high signal variation in DDA acquisition, the variation could be largely reduced in SWATH acquisition. ( c ) In solution protocols yield the highest number of peptides suitable for label-free quantification. The number of peptides with a CV
    Reagent Rapigest Sf, supplied by Waters Corporation, used in various techniques. Bioz Stars score: 76/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/reagent rapigest sf/product/Waters Corporation
    Average 76 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    reagent rapigest sf - by Bioz Stars, 2020-02
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    99
    Waters Corporation rapigest sf
    Protein extraction using the acid-labile detergent <t>RapiGest</t> ™ SF. Protein was extracted with the acid-labile detergent RapiGest ™ (sample “Rapigest”). (A) Workflow and names of resulting samples and approximate duration of
    Rapigest Sf, supplied by Waters Corporation, used in various techniques. Bioz Stars score: 99/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rapigest sf/product/Waters Corporation
    Average 99 stars, based on 87 article reviews
    Price from $9.99 to $1999.99
    rapigest sf - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

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    In solution digestion leads to stable results in label-free proteomics. ( a ) The distribution maximum of coefficients of variation (CV) of the selected protocols varies between 0.075 and 0.2. CV values obtained for protocol triplicates are shown as two-dimensional distribution histograms (‘violin plots’). Quantification in DDA experiments was consistent over the dynamic range, as CV values only marginally changed when filtering by peptides according to their abundance (80%, 60%, 40% or 20%). CV likelihood maxima of all protocols were below 20%, while RapidACN led to the most reproducible results (CV = 7%) (n = 3). ( b ) Stability in a quantification experiment is improved by data-independent acquisition. CV values for the same set of peptides measured with SWATH and DDA using the RapiGest protocol, as shown in a two-dimensional distribution histogram. Whereas there was a high signal variation in DDA acquisition, the variation could be largely reduced in SWATH acquisition. ( c ) In solution protocols yield the highest number of peptides suitable for label-free quantification. The number of peptides with a CV

    Journal: F1000Research

    Article Title: The beauty of being (label)-free: sample preparation methods for SWATH-MS and next-generation targeted proteomics

    doi: 10.12688/f1000research.2-272.v2

    Figure Lengend Snippet: In solution digestion leads to stable results in label-free proteomics. ( a ) The distribution maximum of coefficients of variation (CV) of the selected protocols varies between 0.075 and 0.2. CV values obtained for protocol triplicates are shown as two-dimensional distribution histograms (‘violin plots’). Quantification in DDA experiments was consistent over the dynamic range, as CV values only marginally changed when filtering by peptides according to their abundance (80%, 60%, 40% or 20%). CV likelihood maxima of all protocols were below 20%, while RapidACN led to the most reproducible results (CV = 7%) (n = 3). ( b ) Stability in a quantification experiment is improved by data-independent acquisition. CV values for the same set of peptides measured with SWATH and DDA using the RapiGest protocol, as shown in a two-dimensional distribution histogram. Whereas there was a high signal variation in DDA acquisition, the variation could be largely reduced in SWATH acquisition. ( c ) In solution protocols yield the highest number of peptides suitable for label-free quantification. The number of peptides with a CV

    Article Snippet: In solution digestion The first method tested ( ) is based upon the commercial reagent RapiGest SF (3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (Waters)), an anionic detergent which is depleted from the sample through acidic cleavage.

    Techniques:

    Protocols cover cellular compartments differently. ( a ) RapiGest and eFASP cover a unique space in the proteome. Identified proteins were visualised in a Venn diagram, excluding the in gel protocols. The RapiGest procedure yielded most unique IDs, followed by eFASP and RapidACN (n = 3). ( b ) SDS-containing protocols are best suited for the extraction of membrane proteins. For the analysis of annotated functions in each protocol, selected GO terms were expressed as percentages of identified proteins. While cytosolic proteins were not enriched in any protocol, membrane proteins were preferentially detected in the SDS-containing protocols. (n = 3, Error bars = +/- S.D.) ( c ) Filter-aided sample preparations yield a balanced representation of the proteome. The identified proteins were plotted against the percentage of proteins annotated by the GO term cytosol, in order to illustrate the similarity of extraction properties. The protocol properties required for efficient extraction of membrane and nuclear proteins is inversely correlated with the extraction efficiency for cytosolic proteins, while there is a positive correlation with ribosomal proteins.

    Journal: F1000Research

    Article Title: The beauty of being (label)-free: sample preparation methods for SWATH-MS and next-generation targeted proteomics

    doi: 10.12688/f1000research.2-272.v2

    Figure Lengend Snippet: Protocols cover cellular compartments differently. ( a ) RapiGest and eFASP cover a unique space in the proteome. Identified proteins were visualised in a Venn diagram, excluding the in gel protocols. The RapiGest procedure yielded most unique IDs, followed by eFASP and RapidACN (n = 3). ( b ) SDS-containing protocols are best suited for the extraction of membrane proteins. For the analysis of annotated functions in each protocol, selected GO terms were expressed as percentages of identified proteins. While cytosolic proteins were not enriched in any protocol, membrane proteins were preferentially detected in the SDS-containing protocols. (n = 3, Error bars = +/- S.D.) ( c ) Filter-aided sample preparations yield a balanced representation of the proteome. The identified proteins were plotted against the percentage of proteins annotated by the GO term cytosol, in order to illustrate the similarity of extraction properties. The protocol properties required for efficient extraction of membrane and nuclear proteins is inversely correlated with the extraction efficiency for cytosolic proteins, while there is a positive correlation with ribosomal proteins.

    Article Snippet: In solution digestion The first method tested ( ) is based upon the commercial reagent RapiGest SF (3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (Waters)), an anionic detergent which is depleted from the sample through acidic cleavage.

    Techniques:

    Protein identification in label-free sample preparations. ( a ) Proteolytic digestion efficiencies. Trypsin or Lys-C/trypsin (FASP) digestion efficiencies expressed as relative occurrence of spectra that could be assigned miscleaved peptides (n = 3). ( b ) Amino acid specificity of proteolytic digestion. Relative occurrence of identified peptides with C-terminal lysine or arginine, compared to the average frequency of these amino acids across all individual proteins identified (n = 3, Error bars = +/- S.D.) ( c ) Identified peptides differ per protocol, and correlate with the total peak area as recorded in a DDA experiment. 18 samples derived from the same yeast culture were processed with six protocols in triplicates, and analyzed on a TripleTOF5600 instrument. The number of identified peptides correlates with the total peak area recorded, and indicates the highest identification rate in in solution digests, followed by filter-aided , and in gel procedures. ( d ) Detection of proteins by DDA or SWATH in a label-free experiment. Samples were analyzed in triplicates both for DDA and SWATH acquisition on a TripleTOF5600 instrument, data was searched using paragon (DDA), and Spectronaut (SWATH). SWATH increased the number of detectable proteins in combination with the in solution protocols. In solution protocols RapidACN and RapiGest led to the detection of up to 1000 proteins in single injections, followed by FASP and eFASP, which gave rise to between 250 and 750 proteins, and in gel injections that yielded 300 proteins IDs. Inset: A comparison of protein IDs from the TripleTOF and QExactive platforms shows a linear correlation for the protocols investigated. Data was searched using Mascot (n = 3, Error bars = +/- S.D.).

    Journal: F1000Research

    Article Title: The beauty of being (label)-free: sample preparation methods for SWATH-MS and next-generation targeted proteomics

    doi: 10.12688/f1000research.2-272.v2

    Figure Lengend Snippet: Protein identification in label-free sample preparations. ( a ) Proteolytic digestion efficiencies. Trypsin or Lys-C/trypsin (FASP) digestion efficiencies expressed as relative occurrence of spectra that could be assigned miscleaved peptides (n = 3). ( b ) Amino acid specificity of proteolytic digestion. Relative occurrence of identified peptides with C-terminal lysine or arginine, compared to the average frequency of these amino acids across all individual proteins identified (n = 3, Error bars = +/- S.D.) ( c ) Identified peptides differ per protocol, and correlate with the total peak area as recorded in a DDA experiment. 18 samples derived from the same yeast culture were processed with six protocols in triplicates, and analyzed on a TripleTOF5600 instrument. The number of identified peptides correlates with the total peak area recorded, and indicates the highest identification rate in in solution digests, followed by filter-aided , and in gel procedures. ( d ) Detection of proteins by DDA or SWATH in a label-free experiment. Samples were analyzed in triplicates both for DDA and SWATH acquisition on a TripleTOF5600 instrument, data was searched using paragon (DDA), and Spectronaut (SWATH). SWATH increased the number of detectable proteins in combination with the in solution protocols. In solution protocols RapidACN and RapiGest led to the detection of up to 1000 proteins in single injections, followed by FASP and eFASP, which gave rise to between 250 and 750 proteins, and in gel injections that yielded 300 proteins IDs. Inset: A comparison of protein IDs from the TripleTOF and QExactive platforms shows a linear correlation for the protocols investigated. Data was searched using Mascot (n = 3, Error bars = +/- S.D.).

    Article Snippet: In solution digestion The first method tested ( ) is based upon the commercial reagent RapiGest SF (3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (Waters)), an anionic detergent which is depleted from the sample through acidic cleavage.

    Techniques: Derivative Assay

    Characteristics of label-free sample preparation methods. Left panel: Schematic overview of the different steps in an LC-MS/MS sample preparation method. Right panel: Main characteristics of the protocols compared in this study. Detailled protocols are given in the Supplementary material . Supplementary protocol 1 : In gel/SDS; 2: In gel/ABC; 3: FASP; 4: eFASP; 5: RapiGest; 6: RapidACN.

    Journal: F1000Research

    Article Title: The beauty of being (label)-free: sample preparation methods for SWATH-MS and next-generation targeted proteomics

    doi: 10.12688/f1000research.2-272.v2

    Figure Lengend Snippet: Characteristics of label-free sample preparation methods. Left panel: Schematic overview of the different steps in an LC-MS/MS sample preparation method. Right panel: Main characteristics of the protocols compared in this study. Detailled protocols are given in the Supplementary material . Supplementary protocol 1 : In gel/SDS; 2: In gel/ABC; 3: FASP; 4: eFASP; 5: RapiGest; 6: RapidACN.

    Article Snippet: In solution digestion The first method tested ( ) is based upon the commercial reagent RapiGest SF (3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (Waters)), an anionic detergent which is depleted from the sample through acidic cleavage.

    Techniques: Sample Prep, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Bias of protein size and pI in sample preparation. ( a ) The spectrum of protein sizes is well covered for all protocols examined. The number of identified proteins was plotted against the theoretical molecular weight (MW) for each protocol investigated. Although some protocols yielded higher identifications than others, the MW range was highly reproducible for all of them when comparing against the whole yeast proteome. ( b ) Different representations of protein charges. When comparing to the distribution of theoretical protein pI values of the whole proteome, all investigated protocols showed an under-representation of proteins with a pI of 10. When expressing the total deviation as deviation score d, RapiGest, FASP and RapidACN score best. The d values were calculated as the sum of all differences in % compared to the theoretical proteome occurrence multiplied by 0.1.

    Journal: F1000Research

    Article Title: The beauty of being (label)-free: sample preparation methods for SWATH-MS and next-generation targeted proteomics

    doi: 10.12688/f1000research.2-272.v2

    Figure Lengend Snippet: Bias of protein size and pI in sample preparation. ( a ) The spectrum of protein sizes is well covered for all protocols examined. The number of identified proteins was plotted against the theoretical molecular weight (MW) for each protocol investigated. Although some protocols yielded higher identifications than others, the MW range was highly reproducible for all of them when comparing against the whole yeast proteome. ( b ) Different representations of protein charges. When comparing to the distribution of theoretical protein pI values of the whole proteome, all investigated protocols showed an under-representation of proteins with a pI of 10. When expressing the total deviation as deviation score d, RapiGest, FASP and RapidACN score best. The d values were calculated as the sum of all differences in % compared to the theoretical proteome occurrence multiplied by 0.1.

    Article Snippet: In solution digestion The first method tested ( ) is based upon the commercial reagent RapiGest SF (3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate (Waters)), an anionic detergent which is depleted from the sample through acidic cleavage.

    Techniques: Sample Prep, Molecular Weight, Expressing

    Protein extraction using the acid-labile detergent RapiGest ™ SF. Protein was extracted with the acid-labile detergent RapiGest ™ (sample “Rapigest”). (A) Workflow and names of resulting samples and approximate duration of

    Journal: Methods in enzymology

    Article Title: Sample Preparation for Phosphoproteomic Analysis of Circadian Time Series in Arabidopsis thaliana

    doi: 10.1016/bs.mie.2014.10.022

    Figure Lengend Snippet: Protein extraction using the acid-labile detergent RapiGest ™ SF. Protein was extracted with the acid-labile detergent RapiGest ™ (sample “Rapigest”). (A) Workflow and names of resulting samples and approximate duration of

    Article Snippet: For hydrolysis of RapiGest™ SF, 50 μl of 20% formic acid was added to digested samples “RapiGest” and “No RapiGest pH 8.5”, which lowered the pH below 2.

    Techniques: Protein Extraction

    MALDI MS spectra of ungroomed fingermarks digested with and without RapiGest SF. The proteolytic solution was applied by spray-coat. (a) MALDI MS spectrum generated using a 20 μg/mL trypsin solution containing. RapiGest SF at 0.1% concentration; (b) MALDI MS spectrum generated using a 20 μg/mL trypsin solution with no RapiGest SF. Panels (c) and (d) display a zoom in the regions between 1500-2400 m / z for the spectra in panels (a) and (b) respectively

    Journal: Journal of the American Society for Mass Spectrometry

    Article Title: Alternative Surfactants for Improved Efficiency of In Situ Tryptic Proteolysis of Fingermarks

    doi: 10.1007/s13361-015-1140-z

    Figure Lengend Snippet: MALDI MS spectra of ungroomed fingermarks digested with and without RapiGest SF. The proteolytic solution was applied by spray-coat. (a) MALDI MS spectrum generated using a 20 μg/mL trypsin solution containing. RapiGest SF at 0.1% concentration; (b) MALDI MS spectrum generated using a 20 μg/mL trypsin solution with no RapiGest SF. Panels (c) and (d) display a zoom in the regions between 1500-2400 m / z for the spectra in panels (a) and (b) respectively

    Article Snippet: In particular, there are peptide peaks between m/z 1000 and 2000 Da, present in the RapiGest SF generated peptide profile that are absent in the tryptic digest alone.

    Techniques: Mass Spectrometry, Generated, Concentration Assay