Absolute SILAC for accurate quantitation of proteins in complex mixtures down to the attomole level

被引:154
作者
Hanke, Stefan [1 ]
Besir, Hueseyin [2 ]
Oesterhelt, Dieter [2 ]
Mann, Matthias [1 ]
机构
[1] Max Planck Inst Biochem, Dept Proteom & Signal Transduct, D-82152 Munich, Germany
[2] Max Planck Inst Biochem, Dept Membrane Biochem, D-82152 Munich, Germany
关键词
mass spectrometry; quantitation; quantification; isotope labeling; LC-MS; selected ion monitoring; proteomics; diagnostics; biomarkers; detection limit;
D O I
10.1021/pr7007175
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Mass spectrometry based proteomics can routinely identify hundreds of proteins in a single LC-MS run, and methods have been developed for relative quantitation between differentially treated samples using stable isotopes. However, absolute quantitation has so far required addition of a labeled standard late in the experimental workflow, introducing variability due to sample preparation. Here we present a new variant of the stable isotope labeling by amino acids in cell culture (SILAC) technique termed "Absolute SILAC" that allows accurate quantitation of selected proteins in complex mixtures. SILAC-labeled recombinant proteins produced in vivo or in vitro are used as internal standards, which are directly mixed into lysates of cells or tissues, This minimizes differences in sample processing between the isotope-labeled standard and its endogenous counterpart. We show that it is possible to quantify over several orders of magnitude, even in the background of a whole cell lysate. We furthermore devise a strategy to quantify peptides at or below their signal-to-noise level on hybrid ion trap instruments, shown here for the LTQ-Orbitrap. The data system triggers on peptides of the SILAC-labeled protein, initiating ion collection in a narrow mass range including the endogenous and labeled peptide. This strategy extends the regular detection limit of an LTQ-Orbitrap by at least an order of magnitude and accurately quantifies down to 150 attomole of protein in a Cell lysate without any fractionation prior to LC-MS. We use Absolute SILAC to determine the copy number per cell of growth factor receptor-bound protein 2 (Grb2) in HeLa, HepG2, and C2C12 cells to 5.5 x 10(5), 8.8 x 10(5), and 5.7 x 10(5), respectively, in the exponential growth phase.
引用
收藏
页码:1118 / 1130
页数:13
相关论文
共 46 条
[31]   HOW TO MEASURE AND PREDICT THE MOLAR ABSORPTION-COEFFICIENT OF A PROTEIN [J].
PACE, CN ;
VAJDOS, F ;
FEE, L ;
GRIMSLEY, G ;
GRAY, T .
PROTEIN SCIENCE, 1995, 4 (11) :2411-2423
[32]   Multiplexed absolute quantification for proteomics using concatenated signature peptides encoded by QconCAT genes [J].
Pratt, Julie M. ;
Simpson, Deborah M. ;
Doherty, Mary K. ;
Rivers, Jenny ;
Gaskell, Simon J. ;
Beynon, Robert J. .
NATURE PROTOCOLS, 2006, 1 (02) :1029-1043
[33]   Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics [J].
Rappsilber, J ;
Ishihama, Y ;
Mann, M .
ANALYTICAL CHEMISTRY, 2003, 75 (03) :663-670
[34]   Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips [J].
Rappsilber, Juri ;
Mann, Matthias ;
Ishihama, Yasushi .
NATURE PROTOCOLS, 2007, 2 (08) :1896-1906
[35]   Absolute multiplexed quantitative analysis of protein expression during muscle development using QconCAT [J].
Rivers, Jenny ;
Simpson, Deborah M. ;
Robertson, Duncan H. L. ;
Gaskell, Simon J. ;
Beynon, Robert J. .
MOLECULAR & CELLULAR PROTEOMICS, 2007, 6 (08) :1416-1427
[36]   Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents [J].
Ross, PL ;
Huang, YLN ;
Marchese, JN ;
Williamson, B ;
Parker, K ;
Hattan, S ;
Khainovski, N ;
Pillai, S ;
Dey, S ;
Daniels, S ;
Purkayastha, S ;
Juhasz, P ;
Martin, S ;
Bartlet-Jones, M ;
He, F ;
Jacobson, A ;
Pappin, DJ .
MOLECULAR & CELLULAR PROTEOMICS, 2004, 3 (12) :1154-1169
[37]   A novel strategy for quantitative proteornics using isotope-coded protein labels [J].
Schmidt, A ;
Kellermann, J ;
Lottspeich, F .
PROTEOMICS, 2005, 5 (01) :4-15
[38]   A novel proteomic screen for peptide-protein interactions [J].
Schulze, WX ;
Mann, M .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2004, 279 (11) :10756-10764
[39]   Global differential non-gel proteomics by quantitative and stable labeling of tryptic peptides with oxygen-18 [J].
Staes, A ;
Demol, H ;
Van Damme, J ;
Martens, L ;
Vandekerckhove, J ;
Gevaert, K .
JOURNAL OF PROTEOME RESEARCH, 2004, 3 (04) :786-791
[40]   High sensitivity detection of plasma proteins by multiple reaction monitoring of N-glycosites [J].
Stahl-Zeng, Jianru ;
Lange, Vinzenz ;
Ossola, Reto ;
Eckhardt, Katrin ;
Krek, Wilhelm ;
Aebersold, Ruedi ;
Domon, Bruno .
MOLECULAR & CELLULAR PROTEOMICS, 2007, 6 (10) :1809-1817