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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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 "Microsampling Capillary Electrophoresis Mass Spectrometry Enables Single-cell Proteomics in Complex Tissues: Developing Cell Clones in Live Xenopus laevis and Zebrafish Embryos"

Article Title: Microsampling Capillary Electrophoresis Mass Spectrometry Enables Single-cell Proteomics in Complex Tissues: Developing Cell Clones in Live Xenopus laevis and Zebrafish Embryos

Journal: Analytical chemistry

doi: 10.1021/acs.analchem.9b00345

Benchmarking and optimization of technology performance against whole-cell dissection. (A) Comparison of the number of identified protein groups between microprobe (μP) sampling in 50 mM ammonium bicarbonate (AmBic) containing 10% (v/v) acetonitrile (ACN), 0.05% (v/v) RapiGest, and no additive (AmBic) vs. whole-cell dissection in (Diss). (B) Comparison of quantification based on the number of quantified proteins ( left panel ) and repeatability (relative standard deviation, STD, right panel for quantification. The reproducibility of the CE-MS platform is shown for reference (“CE-MS”). Box-whisker plots comparing expression of representative proteins. Key: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.
Figure Legend Snippet: Benchmarking and optimization of technology performance against whole-cell dissection. (A) Comparison of the number of identified protein groups between microprobe (μP) sampling in 50 mM ammonium bicarbonate (AmBic) containing 10% (v/v) acetonitrile (ACN), 0.05% (v/v) RapiGest, and no additive (AmBic) vs. whole-cell dissection in (Diss). (B) Comparison of quantification based on the number of quantified proteins ( left panel ) and repeatability (relative standard deviation, STD, right panel for quantification. The reproducibility of the CE-MS platform is shown for reference (“CE-MS”). Box-whisker plots comparing expression of representative proteins. Key: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Techniques Used: Dissection, Sampling, Standard Deviation, Whisker Assay, Expressing

3) 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

4) 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

5) 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

6) 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

7) 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

8) 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

9) 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

10) 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

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).

Modification:

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).

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 . ..

Dissection:

Article Title: Microsampling Capillary Electrophoresis Mass Spectrometry Enables Single-cell Proteomics in Complex Tissues: Developing Cell Clones in Live Xenopus laevis and Zebrafish Embryos
Article Snippet: .. – To enhance identification and quantification, different buffers were tested to extract and digest proteins: 50 mM ammonium bicarbonate (AmBic) containing no additive, 10% ACN, or 0.05% RapiGest (Waters Corp.) were compared to the reference approach, viz. whole-cell dissection with digestion in 1% SDS . ..

Mass Spectrometry:

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. ..

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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: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 2 article reviews
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    92
    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: 92/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Waters Corporation surfactant rapigest
    (A‐C) Quality control and label free quantitative analysis comparison between GnHCl, <t>RapiGest™,</t> GnHCl and Rapigest™, and GnHCl followed by RapiGest™ using Progenesis QI software. The variation in percentage of all peptide ions (A), number of peptides (B) and proteins (C) was presented for each extraction method. (D) PCA plot of all methods, GnHCl followed by RapiGest™ samples grouped closer together. (E‐H) Significantly abundant proteins (fold change > 2 and p
    Surfactant Rapigest, supplied by Waters Corporation, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 88 stars, based on 3 article reviews
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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

    (A‐C) Quality control and label free quantitative analysis comparison between GnHCl, RapiGest™, GnHCl and Rapigest™, and GnHCl followed by RapiGest™ using Progenesis QI software. The variation in percentage of all peptide ions (A), number of peptides (B) and proteins (C) was presented for each extraction method. (D) PCA plot of all methods, GnHCl followed by RapiGest™ samples grouped closer together. (E‐H) Significantly abundant proteins (fold change > 2 and p

    Journal: Proteomics

    Article Title: Comparison between chaotropic and detergent‐based sample preparation workflow in tendon for mass spectrometry analysis

    doi: 10.1002/pmic.201700018

    Figure Lengend Snippet: (A‐C) Quality control and label free quantitative analysis comparison between GnHCl, RapiGest™, GnHCl and Rapigest™, and GnHCl followed by RapiGest™ using Progenesis QI software. The variation in percentage of all peptide ions (A), number of peptides (B) and proteins (C) was presented for each extraction method. (D) PCA plot of all methods, GnHCl followed by RapiGest™ samples grouped closer together. (E‐H) Significantly abundant proteins (fold change > 2 and p

    Article Snippet: Another extraction technique that has recently been used in tendon proteomic studies is the surfactant RapiGest™ (Waters) , , which was shown to increase protein identification , .

    Techniques: Software

    (A) 1D SDS‐PAGE analysis of the protein profiles of GnHCl, RapiGest™, GnHCl and RapiGest™, and GnHCl followed by RapiGest™ extraction methods. (B) Protein concentration yielded with the different extraction methods. Values are mean and error bars represent SD, * p

    Journal: Proteomics

    Article Title: Comparison between chaotropic and detergent‐based sample preparation workflow in tendon for mass spectrometry analysis

    doi: 10.1002/pmic.201700018

    Figure Lengend Snippet: (A) 1D SDS‐PAGE analysis of the protein profiles of GnHCl, RapiGest™, GnHCl and RapiGest™, and GnHCl followed by RapiGest™ extraction methods. (B) Protein concentration yielded with the different extraction methods. Values are mean and error bars represent SD, * p

    Article Snippet: Another extraction technique that has recently been used in tendon proteomic studies is the surfactant RapiGest™ (Waters) , , which was shown to increase protein identification , .

    Techniques: SDS Page, Protein Concentration

    (A) Venn diagram of GnHCl, urea, and RapiGest™ extraction methods. Total number, common, and unique proteins identified following MS. All identified proteins in each method are can be found in Supporting Information Table 1. (B) Schematic workflow of follow up experiment using the chaotropic agent GnHCl, the surfactant RapiGest™, a combination of GnHCl and RapiGest™, and a combination of GnHCl followed by RapiGest™ extraction on the insoluble pellet.

    Journal: Proteomics

    Article Title: Comparison between chaotropic and detergent‐based sample preparation workflow in tendon for mass spectrometry analysis

    doi: 10.1002/pmic.201700018

    Figure Lengend Snippet: (A) Venn diagram of GnHCl, urea, and RapiGest™ extraction methods. Total number, common, and unique proteins identified following MS. All identified proteins in each method are can be found in Supporting Information Table 1. (B) Schematic workflow of follow up experiment using the chaotropic agent GnHCl, the surfactant RapiGest™, a combination of GnHCl and RapiGest™, and a combination of GnHCl followed by RapiGest™ extraction on the insoluble pellet.

    Article Snippet: Another extraction technique that has recently been used in tendon proteomic studies is the surfactant RapiGest™ (Waters) , , which was shown to increase protein identification , .

    Techniques: Mass Spectrometry

    (A) The volcano plot demonstrates all differentially abundant proteins between GnHCl and GnHCl followed by RapiGest™ (fold change > 2 and p

    Journal: Proteomics

    Article Title: Comparison between chaotropic and detergent‐based sample preparation workflow in tendon for mass spectrometry analysis

    doi: 10.1002/pmic.201700018

    Figure Lengend Snippet: (A) The volcano plot demonstrates all differentially abundant proteins between GnHCl and GnHCl followed by RapiGest™ (fold change > 2 and p

    Article Snippet: Another extraction technique that has recently been used in tendon proteomic studies is the surfactant RapiGest™ (Waters) , , which was shown to increase protein identification , .

    Techniques: