Table I

Summary of potential peptide and protein identifications electronically filtered at varying degrees of confidence

Lipid rafts were isolated from control and stimulated Jurkat T cells and proteins subjected to ICAT labeling and ultimately μLC-MS/MS analysis, as summarized in Fig. 1 and described under “Experimental Procedures.” Two iterations of this procedure were performed. MS/MS data were searched using SEQUEST™ software against a human protein sequence database. Three separate datasets were compiled: the avidin-affinity eluate fractions (ICAT-labeled Cys-containing peptides) from the two iterations of the experiment (ICAT 1 and ICAT 2), and the avidin-affinity flow-through fractions of the first ICAT experiment (Flow-through 1). The resultant SEQUEST™ output files generated within each dataset were automatically modeled using PeptideProphet™, then manually sorted and filtered. Different computed probability (pcomp) thresholds were set to filter each dataset using INTERACT, which reported the total number of peptide identifications (which includes redundant identifications of the same sequence), the total number of unique peptide sequences identified, the total number of proteins (database entries) these corresponded to, and the number of these which were identified by only a single peptide (single hits). This table lists these numbers for minimum peptide pcomp thresholds of 0.95, 0.9, 0.7, and 0.5 for each of the three datasets. Additional filtering with INTERACT was performed to obtain the same numbers for only Cys-containing peptides, given in parentheses (+C) for the ICAT 1 and 2 datasets. Finally, the number of unique peptides identified is also given as a percentage of total peptide matches, and the number of single hit proteins as a percentage of total proteins identified.

pcomp ≥ 0.95 (+C)pcomp ≥ 0.9 (+C)pcomp ≥ 0.7 (+C)pcomp ≥ 0.5 (+C)
ICAT 1 SEQUEST output files: 38,881Peptides1,322 (1226)1,378 (1.270)1,486 (1,356)1,532 (1,387)
Unique593 (529)610 (538)654 (568)679 (583)
% Unique44.8% (43.1%)44.3% (42.4%)44.0%(41.9%)44.3% (42.0%)
Proteins312 (294)321 (299)343 (311)362 (322)
Single hits110 (103)114 (104)126 (107)143 (117)
% Single hits35.3% (35.0%)35.5% (34.8%)36.7% (34.4%)39.5% (36.3%)
ICAT 2 SEQUEST output files: 14,087Peptides869 (852)912 (893)961 (936)983 (955)
Unique335 (320)348 (332)370 (350)382 (360)
% Unique38.6% (37.6%)38.2% (37.2%)38.5% (37.4%)38.9% (37.7%)
Proteins228 (221)236 (228)250 (240)259 (248)
Single hits97 (94)98 (94)108 (103)115 (109)
% Single hits42.5% (42.5%)41.5% (41.2%)43.2% (42.9%)44.4% (44.0%)
Flow-through 1 SEQUEST output files: 48,831Peptides4,1134,3904,8515,152
Unique1,5321,6071,7481,843
% Unique37.2%36.3%36.0%35.8%
Proteins565592644702
Single hits230247282334
% Single hits40.7%41.7%43.8%47.6%