Large-Scale Proteomic Analysis Using 2D-LE

For large scale global analysis, HeLa S3 cells were prefractionated using custom 2D-LE platform, comprised of sIEF coupled to multiplexed GELFrEE^{12 (link),13 (link)}. HeLa S3, H1299, B16F10 cells, and mitochondrial membrane proteins were also fractionated using the custom GELFrEE^{13 (link)} device alone (no sIEF). After separation, detergent and salt were removed, and the fractions were injected into nanocapillary RPLC columns for elution into a 12 Tesla LTQ FTMS for online detection and fragmentation^{14 (link),15 (link)}. The MS RAW files were processed with in-house software called crawler to assign masses. Using this program, determination of both the intact masses and the corresponding fragment masses were performed and these data were searched against a human proteome database. Extensive statistical workups were also performed using several FDR estimation approaches (with decoy databases both concatenated and not). A final q-value procedure is described in detail (Methods), with the data above reported using a 5% instantaneous FDR (i.e., q-value) cutoff at the protein level (Supplementary Fig. 14).