Analytical characteristics of cleavable isotope-coded affinity tag-LC-tandem mass spectrometry for quantitative proteomic studies.
Quantitative proteomic studies using cleavable isotope-coded affinity tags (cICAT) in concert with tandem mass spectrometry (MS/MS) permit unbiased comparisons between biologically distinct samples. We sought to determine the analytical characteristics of cICAT-based studies by examining the cumulative results of multiple, separate cICAT-based experiments involving human lymphoma-derived cells. We found that the number of identified proteins increased with larger numbers of fractions analyzed. The majority of proteins were identified by single peptides. Only 24 to 41% of the peptides contained cysteine residues, but 85% of the cysteine-containing peptides yielded quantification data. Approximately 28% of all identified proteins yielded quantification data, with 57% of these being differentially expressed by at least 1.5-fold. The quantification ratios of peptides for proteins with multiple quantified peptides were concordant in trend in 87% of instances. cICAT-labeled peptides identified proteins in all subcellular compartments without significant bias. Analysis of the flow-through fraction did not increase the number of peptides identified per protein. Our studies indicate that cICAT-LC-MS/MS yields quantifications primarily based on single peptides, and analysis of flow-through peptides does not contribute signifi-cantly to the results. Nevertheless, identifications based on single cICAT-labeled peptides with tryptic ends provide sufficiently reliable protein identifications and quantification information in cICAT-LC-MS/MS-based proteomic studies.
Vaughn
Cecily P
CP
ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA.
Crockett
David K
DK
Lim
Megan S
MS
Elenitoba-Johnson
Kojo S J
KS
D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D008223:Lymphoma / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000378:metabolism*; D020543:Proteome; D040901:Proteomics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013347:Subcellular Fractions
D010455:Peptides; D011506:Proteins; D020543:Proteome