Quantitative Proteomic Approaches in Mouse: Stable Isotope Incorporation by Metabolic (SILAC) or Chemical Labeling (Reductive Dimethylation) Combined with High‐Resolution Mass Spectrometry

Anja M. Billing1, Hisham Ben Hamidane1, Johannes Graumann1

1 Weill Cornell Medical College in Qatar, Doha, Qatar
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo140156
Online Posting Date:  March, 2015
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Mass spectrometry–based quantitative proteomics is a powerful method for in‐depth exploration of protein expression, allowing researchers to probe its regulation and study signal‐transduction networks, protein turnover, secretion, and spatial distribution, as well as post‐translational modification and protein‐protein interaction, on a large scale. Precise protein quantitation may be achieved by incorporation of stable isotopes, which introduce a mass shift detectable by mass spectrometry, allowing multiplexing of several samples and therefore relative quantification. Stable isotope incorporation into proteins or peptides can be attained either by metabolic labeling (e.g., SILAC) or by chemical labeling (e.g., reductive dimethylation). Both labeling approaches are presented here. They are straightforward and robust and can be applied to murine samples. While both SILAC and reductive dimethylation offer similar multiplexing capabilities and quantitative accuracy, reductive dimethylation is more versatile and can be used with any sample type. © 2015 by John Wiley & Sons, Inc.

Keywords: SILAC; reductive dimethylation; mass spectrometry; quantitative proteomics; tissue; mouse

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Stable Isotope Labeling of Amino Acids (SILAC) in Cell Culture
  • Basic Protocol 2: Reductive Dimethyl Labeling
  • Support Protocol 1: Sample Preparation, Methanol‐Chloroform Precipitation, and In‐Solution Digest
  • Support Protocol 2: Desalting Samples on Oligo R3 Columns
  • Support Protocol 3: Peptide Fractionation with In‐Solution Isoelectric Focusing
  • Support Protocol 4: Desalting by Reversed‐Phase C18 StageTip
  • Support Protocol 5: High‐Performance Liquid Chromatography (HPLC) and Mass Spectrometry (MS): LC‐MS
  • Support Protocol 6: Data Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Stable Isotope Labeling of Amino Acids (SILAC) in Cell Culture

  Materials
  • Cells of interest
  • Items needed to prepare arginine‐ and lysine‐free medium for SILAC (choose according to appropriate basal medium for cells of interest):
    • DMEM, low‐glucose, with L‐glutamine, without arginine, lysine (powder; US Biological, cat. no. D9800‐06)
    • DMEM high‐glucose, with L‐glutamine, without arginine, lysine (powder; US Biological, cat. no. D9803‐07B)
    • DMEM Ham's F‐12 1:1 without arginine, lysine (powder; US Biological, cat. no. D9811‐15C)
    • Ham's F12 medium without arginine, lysine (Thermo Scientific, cat. no. 88424)
    • RPMI 1640 medium with L‐glutamine, HEPES; without arginine, lysine (powder; US Biological, cat. no. R8998‐01)
    • IMDM medium without arginine, lysine (Thermo Scientific, cat. no. 88423)
    • mTeSR1 medium without arginine, lysine (StemCell Technologies, custom made)
    • Arginine and lysine‐free serum for SILAC: dialyzed FBS for SILAC (Thermo Scientific, cat. no. 88440)
  • Light labels:
    • Arginine‐0 (Sigma, cat. no. A5006‐100 G, L‐arginine)
    • Lysine‐0 (Sigma, cat. no. L5501‐100 G, L‐lysine)
  • Medium labels:
    • Arginine‐6 (Cambridge Isotope Laboratories, cat. no. CLM‐2265‐H‐PK, 13C 6‐L‐arginine)
    • Lysine‐4 (Cambridge Isotope Laboratories, cat. no. DLM‐2640‐PK, D 4‐L‐lysine)
  • Heavy labels:
    • Arginine‐10 (Cambridge Isotope Laboratories, cat. no. CNLM‐539‐H‐PK, – 13C 615N 4‐L‐arginine)
    • Lysine‐8 (Cambridge Isotope Laboratories, cat. no. CNLM‐291‐H‐PK, 13C 615N 2‐L‐lysine)

Basic Protocol 2: Reductive Dimethyl Labeling

  Materials
  • Peptide samples of interest
  • Formaldehyde (CH 2O; 37%; Sigma‐Aldrich, cat. no. 252549‐100 ml)
  • Formaldehyde‐d 2 (CD 2O; ∼20 wt.% in D 2O; 98 atom% D; Sigma‐Aldrich, cat. no. 492620‐20 G)
  • Formaldehyde‐13C‐d 2 (13CD 2O; 20 wt.% in D 2O; 98 atom% D; 99 atom% 13C; Sigma‐Aldrich, cat.nr. 596388)
  • HPLC‐grade light water (CHROMASOLV Plus for HPLC; Sigma‐Aldrich, cat. no. 34877)
  • Deuterium oxide (D 2O; heavy water; 99.98 atom% ± 0.01 atom% D; Sigma‐Aldrich, cat. no. 364312)
  • Sodium cyanoborohydride (NaBH 3CN; Sigma‐Aldrich, cat. no. 156159)
  • Sodium cyanoborodeuteride (NaBD 3CN; Santa Cruz Biotechnology, cat. no. sc‐258163)
  • 25% ammonia (EMD Millipore Chemicals, cat. no. 105428)
  • Formic acid (98%; EMD Millipore Chemicals, cat. no. FX0440‐7)
  • Centrifuge
NOTE: In case of duplex, use light and heavy labels. Label samples to be mixed in separate tubes.

Support Protocol 1: Sample Preparation, Methanol‐Chloroform Precipitation, and In‐Solution Digest

  Materials
  • Cultured, labeled cells from SILAC or dimethyl labeling experiment (Basic Protocol protocol 11 or protocol 22)
  • 2% SDS lysis buffer (see recipe)
  • DL‐dithiothreitol [DTT; ≥98% (TLC); ≥99.0% (titration) (Sigma‐Aldrich, cat. no. D0632)]
  • Triethyl ammonium bicarbonate (TEAB), 1 M, pH 8.5 (Sigma‐Aldrich, cat. no. 90360‐500 ml)
  • Iodoacetamide (IAA; BioUltra; Sigma‐Aldrich, cat. no. I1149)
  • Methanol, HPLC grade (Sigma‐Aldrich, cat. no. 34860)
  • Chloroform (Sigma‐Aldrich, cat. no. 372978)
  • Urea buffer (see recipe)
  • Bradford or 2D Quant kit for protein determination
  • Endoproteinase Lys‐C, sequencing grade from Lysobacter enzymogenes (Roche, cat. no. 11 420 429 001)
  • Trypsin, sequencing grade, modified (Promega, cat. no. V511A)
  • HPLC‐grade water CHROMASOLV Plus for HPLC (Sigma‐Aldrich, cat. no. 34877)
  • Probe sonicator
  • Safe Lock tubes 1.5 ml, polypropylene, usable up to 30,000 × g (Eppendorf, cat. no. 0030120.086)
  • Safe Lock tubes 2 ml, polypropylene, usable up to 30,000 × g (Eppendorf, cat. no. 0030123.344)
  • 15‐ml centrifuge tubes, polypropylene, usable up to 17,000 × g (VWR, cat. no. 21008‐216)
  • 50‐ml centrifuge tubes, polypropylene, usable up to 20,000 × g (VWR, cat. no. 21008‐242)
  • Centrifuge

Support Protocol 2: Desalting Samples on Oligo R3 Columns

  Materials
  • Oligo R3 reversed‐phase packing (Applied Biosystems, cat. no. 1‐1339‐06)
  • HPLC‐grade acetonitrile, CHROMASOLV Plus for HPLC (Sigma‐Aldrich, cat. no. 270717)Trifluoroacetic acid (TFA; Sigma‐Aldrich, cat. no. T6508) in HPLC‐grade water (CHROMASOLV Plus for HPLC; Sigma‐Aldrich, cat. no. 34877)
  • Protein or peptide sample for desalting (Basic Protocol protocol 11 or protocol 22)
  • B60 buffer: 0.1% (v/v) TFA in 60% (v/v) acetonitrile in HPLC‐grade H 2O
  • C18 Empore high‐performance extraction disks (Octadecyl; 3 M; Dr. Maisch GmBH, cat. no. 2215)
  • 200‐μl (P‐200) pipet tips
  • StageTip centrifuge (Sonation, ZF‐MO‐230 V)
  • Vacuum concentrator
  • Additional reagents and equipment for preparation of StageTips ( protocol 6)

Support Protocol 3: Peptide Fractionation with In‐Solution Isoelectric Focusing

  Materials
  • Peptide sample for fractionation: SILAC sample after in‐solution digest ( protocol 3) or dimethyl‐labeled sample after desalting ( protocol 4)
  • 3100 OFFGEL Low Res Kit, pH 3‐10 (Agilent, cat. no. 5188‐6425 (12 fractions; kit includes IPG strips, glycerol, ampholytes, paper pads, fractionation frames, and frame lids)
  • 1.25× OFFGEL stock solution (see recipe)
  • Rehydration solution: 0.56 ml 1.25× OFFGEL stock solution/0.14 ml HPLC‐grade H 2O (per IPG strip, 12‐fraction format)
  • A* buffer: 2% (v/v) acetonitrile/0.1% (w/v) TFA in HPLC‐grade H 2O
  • Immobiline Dry Strips, pH 3‐10, NL, 13 cm (GE Healthcare, cat. no. 17600115; 12 fractions; store at –20°C)
  • 3100 OFFGEL fractionator (Agilent)

Support Protocol 4: Desalting by Reversed‐Phase C18 StageTip

  Materials
  • A* buffer: 2% (v/v) acetonitrile/0.1% (w/v) trifluoroacetic acid (TFA) in HPLC‐grade H 2O
  • A buffer: 0.5% (v/v) acetic acid in HPLC‐grade H 2O
  • C18 Empore High‐Performance Extraction Disks (Octadecyl; 3 M; Dr. Maisch GmBH, Cat.nr. 2215)
  • 200‐μl (P‐200) pipet tips
  • Metal‐hub blunt‐point needle, 17‐G, point style 3 (Hamilton, cat. no. 91017)
  • StageTip centrifuge (Sonation, ZF‐MO‐230 V)

Support Protocol 5: High‐Performance Liquid Chromatography (HPLC) and Mass Spectrometry (MS): LC‐MS

  Materials
  • A buffer: 0.5% (v/v) acetic acid in HPLC‐grade water
  • A* buffer: 2% (v/v) acetonitrile/0.1% (w/v) trifluoroacetic acid (TFA) in HPLC‐grade water
  • Sample‐containing StageTip ( protocol 6)
  • B60 buffer: 60% (v/v) acetonitrile/0.1% (v/v) trifluoroacetic acid (TFA) in HPLC‐grade water
  • Mobile phase A: 0.1% (v/v) formic acid in LC‐MS grade water
  • Mobile phase B: 0.1% (v/v) formic acid in 90:10 acetonitrile:LC‐MS grade water
  • Low‐binding mass spectrometry tubes
  • Vacuum concentrator
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Altelaar, A.F.M., Frese, C.K., Preisinger, C., Hennrich, M.L., Schram, A.W., Timmers, H.T.M., Heck, A.J.R., and Mohammed, S. 2013. Benchmarking stable isotope labeling based quantitative proteomics. J. Proteomics 88:14‐26.
  Bendall, S.C., Hughes, C., Stewart, M.H., Doble, B., Bhatia, M., and Lajoie, G.A. 2008. Prevention of amino acid conversion in SILAC experiments with embryonic stem cells. Mol. Cell. Proteomics MCP 7:1587‐1597.
  Blanc, V., Park, E., Schaefer, S., Miller, M., Lin, Y., Kennedy, S., Billing, A.M., Ben Hamidane, H., Graumann, J., Mortazavi, A., Nadeau, J.H., and Davidson, N.O. 2014. Genome‐wide identification and functional analysis of Apobec‐1 mediated C‐to‐U RNA editing in mouse small intestine and liver. Genome Biol. 15:R79.
  Boersema, P.J., Aye, T.T., van Veen, T.A.B., Heck, A.J.R., and Mohammed, S. 2008. Triplex protein quantification based on stable isotope labeling by peptide dimethylation applied to cell and tissue lysates. Proteomics 8:4624‐4632.
  Boersema, P.J., Raijmakers, R., Lemeer, S., Mohammed, S., and Heck, A.J.R. 2009. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat. Protoc. 4:484‐494.
  Cao, R., Chen, K., Song, Q., Zang, Y., Li, J., Wang, X., Chen, P., and Liang, S. 2012. Quantitative proteomic analysis of membrane proteins involved in astroglial differentiation of neural stem cells by SILAC labeling coupled with LC–MS/MS. J. Proteome Res. 11:829‐838.
  Chen, Y., Colak, G., and Zhao, Y. 2013. SILAC‐based quantification of Sirt1‐responsive lysine acetylome. Methods Mol. Biol. Clifton N.J. 1077:105‐120.
  Cox, J. and Mann, M. 2008. MaxQuant enables high peptide identification rates, individualized p.p.b.‐range mass accuracies and proteome‐wide protein quantification. Nat. Biotechnol. 26:1367‐1372.
  Cox, J., Neuhauser, N., Michalski, A., Scheltema, R.A., Olsen, J.V., and Mann, M. 2011. Andromeda: A peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10:1794‐1805.
  Dayon, L., Hainard, A., Licker, V., Turck, N., Kuhn, K., Hochstrasser, D.F., Burkhard, P.R., and Sanchez, J.‐C. 2008. Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6‐plex isobaric tags. Anal. Chem. 80:2921‐2931.
  de Graaf, E.L., Vermeij, W.P., de Waard, M.C., Rijksen, Y., van der Pluijm, I., Hoogenraad, C.C., Hoeijmakers, J.H.J., Altelaar, A.F.M., and Heck, A.J.R. 2013. Spatio‐temporal analysis of molecular determinants of neuronal degeneration in the aging mouse cerebellum. Mol. Cell. Proteomics MCP 12:1350‐1362.
  Di Palma, S., Raijmakers, R., Heck, A.J.R., and Mohammed, S. 2011. Evaluation of the deuterium isotope effect in zwitterionic hydrophilic interaction liquid chromatography separations for implementation in a quantitative proteomic approach. Anal. Chem. 83:8352‐8356.
  Engholm‐Keller, K., Birck, P., Størling, J., Pociot, F., Mandrup‐Poulsen, T., and Larsen, M.R. 2012. TiSH–a robust and sensitive global phosphoproteomics strategy employing a combination of TiO2, SIMAC, and HILIC. J. Proteomics 75:5749‐5761.
  Fierro‐Monti, I., Racle, J., Hernandez, C., Waridel, P., Hatzimanikatis, V., and Quadroni, M. 2013. A novel pulse‐chase SILAC strategy measures changes in protein decay and synthesis rates induced by perturbation of proteostasis with an Hsp90 Inhibitor. PLoS ONE 8:e80423.
  Geiger, T., Velic, A., Macek, B., Lundberg, E., Kampf, C., Nagaraj, N., Uhlen, M., Cox, J., and Mann, M. 2013. Initial quantitative proteomic map of 28 mouse tissues using the SILAC mouse. Mol. Cell. Proteomics MCP 12:1709‐1722.
  Graumann, J., Hubner, N.C., Kim, J.B., Ko, K., Moser, M., Kumar, C., Cox, J., Schöler, H., and Mann, M. 2008. Stable isotope labeling by amino acids in cell culture (SILAC) and proteome quantitation of mouse embryonic stem cells to a depth of 5,111 proteins. Mol. Cell. Proteomics 7:672‐683.
  Guo, K., Ji, C., and Li, L. 2007. Stable‐Isotope dimethylation labeling combined with LC−ESI MS for quantification of amine‐containing metabolites in biological samples. Anal. Chem. 79:8631‐8638.
  Hsu, J.‐L., Huang, S.‐Y., Chow, N.‐H., and Chen, S.‐H. 2003. Stable‐isotope dimethyl labeling for quantitative proteomics. Anal. Chem. 75:6843‐6852.
  Ji, C., Li, L., Gebre, M., Pasdar, M., and Li, L. 2005. Identification and quantification of differentially expressed proteins in E‐cadherin deficient SCC9 cells and SCC9 transfectants expressing E‐cadherin by dimethyl isotope labeling, LC‐MALDI MS and MS/MS. J. Proteome Res. 4:1419‐1426.
  Krüger, M., Moser, M., Ussar, S., Thievessen, I., Luber, C.A., Forner, F., Schmidt, S., Zanivan, S., Fässler, R., and Mann, M. 2008. SILAC mouse for quantitative proteomics uncovers kindlin‐3 as an essential factor for red blood cell function. Cell 134:353‐364.
  Larance, M., Bailly, A.P., Pourkarimi, E., Hay, R.T., Buchanan, G., Coulthurst, S., Xirodimas, D.P., Gartner, A., and Lamond, A.I. 2011. Stable‐isotope labeling with amino acids in nematodes. Nat. Methods 8:849‐851.
  Liao, L., Park, S.K., Xu, T., Vanderklish, P., and Yates, J.R. 2008. Quantitative proteomic analysis of primary neurons reveals diverse changes in synaptic protein content in fmr1 knockout mice. Proc. Natl. Acad. Sci. U.S.A. 105:15281‐15286.
  Liberski, A.R., Al‐Noubi, M.N., Rahman, Z.H., Halabi, N.M., Dib, S.S., Al‐Mismar, R., Billing, A.M., Krishnankutty, R., Ahmad, F.S., Raynaud, C.M., Rafii, A., Engholm‐Keller, K., and Graumann, J., 2013. Adaptation of a commonly used, chemically defined medium for human embryonic stem cells to stable isotope labeling with amino acids in cell culture. J. Proteome. Res. 12:3233‐3245.
  Mann, M. 2006. Functional and quantitative proteomics using SILAC. Nat. Rev. Mol. Cell Biol. 7:952‐958.
  Marcilla, M., Alpizar, A., Paradela, A., and Albar, J.P. 2011. A systematic approach to assess amino acid conversions in SILAC experiments. Talanta 84:430‐436.
  McAlister, G.C., Huttlin, E.L., Haas, W., Ting, L., Jedrychowski, M.P., Rogers, J.C., Kuhn, K., Pike, I., Grothe, R.A., Blethrow, J.D., and Gygi, S.P. 2012. Increasing the multiplexing capacity of TMT using reporter ion isotopologues with isobaric masses. Anal. Chem. 84:7469‐7478.
  Melo‐Braga, M.N., Schulz, M., Liu, Q., Swistowski, A., Palmisano, G., Engholm‐Keller, K., Jakobsen, L., Zeng, X., and Larsen, M.R. 2014. Comprehensive quantitative comparison of the membrane proteome, phosphoproteome, and sialiome of human embryonic and neural stem cells. Mol. Cell. Proteomics 13:311‐328.
  Mertins, P., Udeshi, N.D., Clauser, K.R., Mani, D.R., Patel, J., Ong, S., Jaffe, J.D., and Carr, S.A. 2012. iTRAQ labeling is superior to mTRAQ for quantitative global proteomics and phosphoproteomics. Mol. Cell. Proteomics MCP 11:M111.014423.
  Monetti, M., Nagaraj, N., Sharma, K., and Mann, M. 2011. Large‐scale phosphosite quantification in tissues by a spike‐in SILAC method. Nat. Methods 8:655‐658.
  Ong, S.‐E., Blagoev, B., Kratchmarova, I., Kristensen, D.B., Steen, H., Pandey, A., and Mann, M. 2002. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1:376‐386.
  Park, S.K., Liao, L., Kim, J.Y., and Yates, J.R. 2009. A computational approach to correct arginine‐to‐proline conversion in quantitative proteomics. Nat. Methods 6:184‐185.
  Pines, A., Kelstrup, C.D., Vrouwe, M.G., Puigvert, J.C., Typas, D., Misovic, B., de Groot, A., von Stechow, L., van de Water, B., Danen, E.H.J., Vrieling, H., Mullenders, L.H., and Olsen, J.V. 2011. Global phosphoproteome profiling reveals unanticipated networks responsive to cisplatin treatment of embryonic stem cells. Mol. Cell. Biol. 31:4964‐4977.
  Rappsilber, J., Ishihama, Y., and Mann, M. 2003. Stop and go extraction tips for matrix‐assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75:663‐670.
  Rayavarapu, S., Coley, W., Cakir, E., Jahnke, V., Takeda, S., Aoki, Y., Grodish‐Dressman, H., Jaiswal, J.K., Hoffman, E.P., Brown, K.J., Hathout, Y., and Nagaraju, K. 2013. Identification of disease specific pathways using in vivo SILAC proteomics in dystrophin deficient mdx mouse. Mol. Cell. Proteomics MCP 12:1061‐1073.
  Robles, M.S., Cox, J., and Mann, M. 2014. In‐vivo quantitative proteomics reveals a key contribution of post‐transcriptional mechanisms to the circadian regulation of liver metabolism. PLoS Genet 10:e1004047.
  Ross, P.L., Huang, Y.N., Marchese, J.N., Williamson, B., Parker, K., Hattan, S., Khainovski, N., Pillai, S., Dey, S., Daniels, S., Daniels, S., Purkayastha, S., Juhasz, P., Martin, S., Bartlet‐Jones, M., He, F., Jacobson, A., and Pappin, D.J. 2004. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine‐reactive isobaric tagging reagents. Mol. Cell. Proteomics MCP 3:1154‐1169.
  Sahasrabuddhe, N.A., Huang, T.‐C., Ahmad, S., Kim, M.‐S., Yang, Y., Ghosh, B., Leach, S.D., Gowda, H., Somani, B.L., Chaerkady, R., and Pandey, A. 2014. Regulation of PPAR‐alpha pathway by Dicer revealed through proteomic analysis. J. Proteomics 108C:306‐315.
  Scholten, A., Mohammed, S., Low, T.Y., Zanivan, S., Veen, T.A.B. van, Delanghe, B., and Heck, A.J.R. 2011. In‐depth quantitative cardiac proteomics combining electron transfer dissociation and the metalloendopeptidase Lys‐N with the SILAC mouse. Mol. Cell. Proteomics 10:O111.008474.
  Stauch, K.L., Purnell, P.R., and Fox, H.S. 2014. Quantitative proteomics of synaptic and nonsynaptic mitochondria: Insights for synaptic mitochondrial vulnerability. J. Proteome Res. 13:2620‐2635.
  Sury, M.D., Chen, J.‐X., and Selbach, M. 2010. The SILAC fly allows for accurate protein quantification in vivo. Mol. Cell. Proteomics 9:2173‐2183.
  Ting, L., Rad, R., Gygi, S.P., and Haas, W. 2011. MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics. Nat. Meth. 8:937‐940.
  Van Hoof, D., Pinkse, M.W.H., Oostwaard, D.W.‐V., Mummery, C.L., Heck, A.J.R., and Krijgsveld, J. 2007. An experimental correction for arginine‐to‐proline conversion artifacts in SILAC‐based quantitative proteomics. Nat. Methods 4:677‐678.
  Walther, D.M. and Mann, M. 2011. Accurate quantification of more than 4000 mouse tissue proteins reveals minimal proteome changes during aging. Mol. Cell. Proteomics MCP 10:M110.004523.
  Wang, F., Blanchard, A.P., Elisma, F., Granger, M., Xu, H., Bennett, S.A.L., Figeys, D., and Zou, H. 2013. Phosphoproteome analysis of an early onset mouse model (TgCRND8) of Alzheimer's disease reveals temporal changes in neuronal and glia signaling pathways. Proteomics 13:1292‐1305.
  Werner, T., Sweetman, G., Savitski, M.F., Mathieson, T., Bantscheff, M., and Savitski, M.M. 2014. Ion coalescence of neutron encoded TMT 10‐plex reporter ions. Anal. Chem. 86:3594‐3601.
  Wessel, D. and Flügge, U.I. 1984. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138:141‐143.
  Wilson‐Grady, J.T., Haas, W., and Gygi, S.P. 2013. Quantitative comparison of the fasted and re‐fed mouse liver phosphoproteomes using lower pH reductive dimethylation. Methods San Diego Calif 61:277‐286.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library