for large scale global analysis , hela s3 cells were prefractionated using custom 2d - le platform , comprised of sief coupled to multiplexed gelfree .
hela s3 , h1299 , b16f10 cells , and mitochondrial membrane proteins were also fractionated using the custom gelfree 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 .
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 .
 a full description of the human proteome relies on the challenging task of detecting mature and changing forms of protein molecules in the body . 
 large scale proteome analysis1 has routinely involved digesting intact proteins followed by inferred protein identification using mass spectrometry ( ms)2 . this  
 bottom up  
 process affords a high number of identifications ( not always unique to a single gene ) . 
 however , complications arise from incomplete or ambiguous2 characterization of alternative splice forms , diverse modifications ( e.g. , acetylation and methylation ) , and endogenous protein cleavages , especially when combinations of these create complex patterns of intact protein isoforms and species3 . 
  top down  interrogation of whole proteins can overcome these problems for individual proteins4,5 , but has not been achieved on a proteome scale due to the lack of intact protein fractionation methods that are well integrated with tandem ms . here 
 we show , using a new four dimensional ( 4d ) separation system , identification of 1,043 gene products from human cells that are dispersed into > 3,000 protein species created by post - translational modification , rna splicing , and proteolysis . 
 the overall system produced > 20-fold increases in both separation power and proteome coverage , enabling the identification of proteins up to 105 kilodaltons and those with up to 11 transmembrane helices . 
 many previously undetected isoforms of endogenous human proteins were mapped , including changes in multiply - modified species in response to accelerated cellular aging ( senescence ) induced by dna damage . integrated with the latest version of the swiss - prot database6 , 
 the data provide precise correlations to individual genes and proof - of - concept for large scale interrogation of whole protein molecules . 
 the technology promises to improve the link between proteomics data and complex phenotypes in basic biology and disease research7 . 