Abstract
Protein expression profiles vary considerably between human cell lines and tissues, which is in part a reflection of their specialized roles within an organism. It is of considerable practical use to establish which proteins constitute the primary components of the respective proteomes. When compiled into databases, such information can facilitate the assessment of selectivity and specificity of a wide range of proteomic experiments. Here we describe the major constituents of proteomes of six human immortalized cell lines. By employing a combination of one-dimensional SDS-PAGE and nanocapillary liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified up to 1785 non-redundant cytoplasmic and nuclear proteins from a single cell line using 50 and 30 microg of total protein from the corresponding fractions. Up to 38 proteins could be identified from a single band in one liquid chromatography-MS/MS experiment. When combined with systematic gridding of gel lanes into 48 slices, a dynamic range for protein identification of approximately 1:2000 can be envisaged for this approach. Identified proteins range from 4-553 kDa in size, cover the pI range between 3.4 and 12.8, and include 255 proteins with predicted transmembrane domains. Repeated analysis of peptides derived from the same gel band showed that the reproducibility of nanocapillary liquid chromatography-MS/MS of such complex mixtures is about 60-70% suggesting that a particular analytical experiment would need to be repeated about three times to arrive at a representative estimate of the set of highly abundant proteins in a given proteome. Given its technical simplicity, sensitivity, and wealth of generated information, we have adopted this experimental approach to characterize every cell line and tissue that is the subject of experimentation in our laboratory. The combined dataset for the six cell lines consists of 2341 non-redundant human proteins and thus constitutes one of the largest collections of human proteomic data published to date.
Highlights
Protein expression profiles vary considerably between human cell lines and tissues, which is in part a reflection of their specialized roles within an organism
Analysis of Core Proteome Datasets Obtained by GeLCMS/mass spectrometry (MS)—Using a simple 1D PAGE separation step prior to nanocapillary LC-MS/MS analysis, we outlined cytoplasmic core proteomes of different human cell lines that are frequently used for a wide range of experimental applications in our and many other laboratories
Up to 1111 non-redundant proteins (991 clusters of 97% sequence identity, HEK293 cytoplasmic) could be identified from 50 g of total protein separated by 1D PAGE followed by slicing gel lanes into 48 bands and measuring tryptic peptides from all 48 gel fractions in triplicate by LCMS/MS
Summary
Protein expression profiles vary considerably between human cell lines and tissues, which is in part a reflection of their specialized roles within an organism. We report on the application of GeLC-MS/MS to the analysis of core proteomes of six human immortalized cell lines as well as on the effect of subcellular fractionation on the total yield of protein identifications.
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