title: Comparison of stable-isotope labeling with amino acids in cell culture and spectral counting for relative quantification of protein expression.
abstract: Protein quantification is one of the principal goals of mass spectrometry (MS)-based proteomics, and many strategies exist to achieve it. Several approaches involve the incorporation of a stable-isotope label using either chemical derivatization, enzymatically catalyzed incorporation of (18)O, or metabolic labeling in a cell or tissue culture. These techniques can be cost or time prohibitive or not amenable to the biological system of interest. Label-free techniques including those utilizing integrated ion abundance and spectral counting offer an alternative to stable-isotope-based methodologies. Herein, we present the comparison of stable-isotope labeling of amino acids in cell culture (SILAC) with spectral counting for the quantification of human embryonic stem cells as they differentiate toward the trophectoderm at three time points. Our spectral counting experimental strategy resulted in the identification of 2641 protein groups across three time points with an average sequence coverage of 30.3%, of which 1837 could be quantified with more than five spectral counts. SILAC quantification was able to identify 1369 protein groups with an average coverage of 24.7%, of which 1027 could be quantified across all time points. Within this context we further explore the capacity of each strategy for proteome coverage, variation in quantification, and the relative sensitivity of each technique to the detection of change in relative protein expression.
authors.affiliation: WM Keck FT-ICR Mass Spectrometry Laboratory, Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Collier
authors.forename: Shan M
authors.initials: SM
authors.lastname: Randall
authors.forename: Prasenjit
authors.initials: P
authors.lastname: Sarkar
authors.forename: Balaji M
authors.initials: BM
authors.lastname: Rao
authors.forename: Ralph A
authors.initials: RA
authors.lastname: Dean
authors.forename: David C
authors.initials: DC
authors.lastname: Muddiman
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D004475:Ectoderm; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D010449:Peptide Mapping; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry; D013997:Time Factors
chemical_list: D000596:Amino Acids; D010446:Peptide Fragments; D011506:Proteins