Journal: Protein Science : A Publication of the Protein Society
Article Title: Human cells for human proteins: Isotope labeling in mammalian cells in suspension for functional NMR studies
doi: 10.1002/pro.70515
Figure Lengend Snippet: Special cases. (a) Methionine labeling in full media by enzymatic removal of intrinsic methionine from a medium with unknown composition, here HEK293 Freestyle medium (GIBCO). On the left, the reaction of the enzyme is shown (for reaction conditions see main text). Note that methanethiol is volatile and thus is continuously removed from the reaction resulting in complete turnover of methionine. In the middle, the methionine signal in the medium is shown at 0, 24 and 48 h after treatment with MGL. To monitor the reaction, the medium was spiked with 1 g/L 13 C‐Met and 13 C‐edited 1D 1 H spectra were recorded. On the right, a 2D [ 13 C, 1 H] HMQC spectrum of 13 C‐Met labeled mEGFP is shown, produced from enzymatically methionine‐free HEK293 Freestyle medium (GIBCO). (b1) Isotope labeling of alanine with inhibition of alanine transaminase to suppress unwanted isotope dilution from pyruvate. On the left, the reaction of alanine transaminase is shown. Note that unlabeled pyruvate is present in rather high concentrations in the cell, thus driving the reaction towards the product alanine. Addition of l ‐cycloserine (red) inhibits this reaction. On the right, a 2D [ 13 C, 1 H]‐XL‐ALSOFAST‐HMQC spectrum of 2 H α , 13 C β ‐Ala labeled mEGFP is shown, which was produced in presence of 50 mg/L of l ‐cycloserine. (b2) Scrambling can also be observed for other amino acids, like in the example of valine labeling of mEGFP shown here. With the standard protocol, scrambling of the labeled valine precursor to leucine is apparent. Addition of an excess of unlabeled leucine (460 mg/L instead of 115 mg/L) changes the balance of the equilibrium reaction and suppresses scrambling. Note that the spectrum on the right hand side (same type as in b1) is plotted at very low contour levels, and it was recorded in detergent‐containing buffer, leading to the strong signal indicated by the dashed horizontal line. (c) When selectively 13 C methyl labeled amino acids are not available, suppression of 1 J CC couplings in u‐ 13 C labeled amino acids yields sharp signals. On the top left, a schematic representation of isoleucine is shown and on the right, the M z ‐excitation profile of an adapted BADCOP1 pulse is shown. The pulse was time‐reversed and the length adjusted in order to selectively invert β and γ1 nuclei, resulting in decoupling of the adjacent γ2 and δ1 nuclei, respectively. (BADCOP1_TR, pulse length: 1700 μs, offset: 16 ppm, simulated in Topspin shapetool). Typical chemical shifts of carbon nuclei are indicated with colored bands (BMRB; Ulrich et al., ). Below 2D [ 13 C, 1 H]‐XL‐ALSOFAST‐HMQC spectra are plotted, showing the methyl region of 13 C‐Ile labeled mEGFP, without (left) and with (right) application of a BADCOP1_TR 180° pulse. A slice through one representative signal is shown in the top right corner of each spectrum and illustrates the suppression of the homonuclear couplings as well as the concurrent ~2‐fold gain in sensitivity. Spectra were recorded on Bruker Avance IIIHD 800 MHz (a) and 900 MHz (b, c) spectrometers equipped with TCI CryoProbes.
Article Snippet: Purified mEGFP obtained in isotope labeled fashion from HEK293 cultures was buffer exchanged to remove traces of detergent using a 3 kDa concentrator (Merck) and 10 μg of mEGFP was subsequently hydrolysed under 6 M HCl with 0.1% w/v phenol steam at 110°C for 24 h before being dried by SpeedVac. (Crabb et al., ) The free amino acids were derivatized (Cohen, ) using the AccQ‐Tag kit (Waters) following the recommendations of the supplier and using an internal standard (MSK‐A2, Cambridge Isotopes Laboratories).
Techniques: Labeling, Produced, Quantitative Proteomics, Inhibition, Isotope Dilution
Addgene inc is a verified supplier 
![Approaches for uniform isotope labeling in mammalian cells in suspension. (a) Microscope images of fluorescent cells from full medium and labeling medium. (b) Yields of <t>mEGFP</t> in mg/L from different media formulations are shown: Full V3 medium (V3, red), V3 ⊖ with yeast extract added back (V3 ⊖ + YE, orange), V3 ⊖ with algal extract (Celtone) added (V3 ⊖ + AE, dark green), V3 ⊖ with delipidated (DL) algal extract added (V3 ⊖ + AE DL , bright green) and FreeStyle medium (blue). The yields of mEGFP are in the mg range because it was expressed as a fusion to β 1 AR and released by protease cleavage. (c–e) 2D TROSY‐HSQC spectra of u‐ 15 N labeled mEGFP produced in <t>stable</t> <t>HEK293</t> GnTI − cells either with (c) 15 N labeled yeast extract or (d) delipidated 13 C, 15 N‐labeled algal extract (ISOGRO, Sigma). All amino acid types were labeled, since identical signals are visible as reported from E. coli expression. Incorporation was 70%, as determined by mass spectrometry. (e and f) [ 15 N, 1 H]‐ and [ 13 C, 1 H]‐HSQC spectra of ILV‐labeled mEGFP produced with E. coli extracts with the same labeling pattern.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9750/pmc12969750/pmc12969750__PRO-35-e70515-g007.jpg)
![Amino acid type‐specific labeling using the 15 N, 13 C, and 2 H isotope labeled amino acids indicated on the right in each spectrum. 2D [ 15 N, 1 H] and [ 13 C, 1 H] correlation spectra of mEGFP produced in stably transfected HEK293 GnTI − cells grown in suspension are shown. See text and Data for exact experimental conditions.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9750/pmc12969750/pmc12969750__PRO-35-e70515-g003.jpg)
![Special cases. (a) Methionine labeling in full media by enzymatic removal of intrinsic methionine from a medium with unknown composition, here HEK293 Freestyle medium (GIBCO). On the left, the reaction of the enzyme is shown (for reaction conditions see main text). Note that methanethiol is volatile and thus is continuously removed from the reaction resulting in complete turnover of methionine. In the middle, the methionine signal in the medium is shown at 0, 24 and 48 h after treatment with MGL. To monitor the reaction, the medium was spiked with 1 g/L 13 C‐Met and 13 C‐edited 1D 1 H spectra were recorded. On the right, a 2D [ 13 C, 1 H] HMQC spectrum of 13 C‐Met labeled mEGFP is shown, produced from enzymatically methionine‐free HEK293 Freestyle medium (GIBCO). (b1) Isotope labeling of alanine with inhibition of alanine transaminase to suppress unwanted isotope dilution from pyruvate. On the left, the reaction of alanine transaminase is shown. Note that unlabeled pyruvate is present in rather high concentrations in the cell, thus driving the reaction towards the product alanine. Addition of l ‐cycloserine (red) inhibits this reaction. On the right, a 2D [ 13 C, 1 H]‐XL‐ALSOFAST‐HMQC spectrum of 2 H α , 13 C β ‐Ala labeled mEGFP is shown, which was produced in presence of 50 mg/L of l ‐cycloserine. (b2) Scrambling can also be observed for other amino acids, like in the example of valine labeling of mEGFP shown here. With the standard protocol, scrambling of the labeled valine precursor to leucine is apparent. Addition of an excess of unlabeled leucine (460 mg/L instead of 115 mg/L) changes the balance of the equilibrium reaction and suppresses scrambling. Note that the spectrum on the right hand side (same type as in b1) is plotted at very low contour levels, and it was recorded in detergent‐containing buffer, leading to the strong signal indicated by the dashed horizontal line. (c) When selectively 13 C methyl labeled amino acids are not available, suppression of 1 J CC couplings in u‐ 13 C labeled amino acids yields sharp signals. On the top left, a schematic representation of isoleucine is shown and on the right, the M z ‐excitation profile of an adapted BADCOP1 pulse is shown. The pulse was time‐reversed and the length adjusted in order to selectively invert β and γ1 nuclei, resulting in decoupling of the adjacent γ2 and δ1 nuclei, respectively. (BADCOP1_TR, pulse length: 1700 μs, offset: 16 ppm, simulated in Topspin shapetool). Typical chemical shifts of carbon nuclei are indicated with colored bands (BMRB; Ulrich et al., ). Below 2D [ 13 C, 1 H]‐XL‐ALSOFAST‐HMQC spectra are plotted, showing the methyl region of 13 C‐Ile labeled mEGFP, without (left) and with (right) application of a BADCOP1_TR 180° pulse. A slice through one representative signal is shown in the top right corner of each spectrum and illustrates the suppression of the homonuclear couplings as well as the concurrent ~2‐fold gain in sensitivity. Spectra were recorded on Bruker Avance IIIHD 800 MHz (a) and 900 MHz (b, c) spectrometers equipped with TCI CryoProbes.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9750/pmc12969750/pmc12969750__PRO-35-e70515-g002.jpg)
![Labeling of methyl groups of Val, Leu, and Ile using 13 C and 2 H isotope labeled precursors indicated on the right in each spectrum. 2D [ 13 C, 1 H] correlation spectra of mEGFP produced in stably transfected HEK293 GnTI − cells are shown. (c) For Ile, only the uniformly 13 C labeled precursor was available, the precursor in gray represents a proposed labeling pattern for similar spectra as in (a) and (b). The strong signal (marked with * in a) stems from detergent present in the buffer. See text and Data for exact experimental conditions.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9750/pmc12969750/pmc12969750__PRO-35-e70515-g005.jpg)