Molecular weight assessment of proteins in total proteome profiles using 1D-PAGE and LC/MS/MS

被引：23

作者：

Ahmad Q.R. ^{[1
]}

Nguyen D.H. ^{[2
]}

Wingerd M.A. ^{[1
]}

Church G.M. ^{[2
,3
]}

Steffen M.A. ^{[1
,4
]}

机构：

[1] Dept. of Genetics and Genomics, Boston University School of Medicine, Boston University, Boston, MA 02118

[2] Dept. of Genetics, Harvard Medical School, Boston, MA 02115

[3] The Lipper Center for Computational Genetics, Harvad Medical School, Boston, MA 02115

[4] Dept. of Biomedical Engineering, Boston University, Boston, MA 02215

来源：

Proteome Science | / 3卷 / 1期

关键词：

Average Molecular Weight; Tryptic Peptide; Intact Protein; High Throughput Manner; Full Length Amino Acid Sequence;

D O I：

10.1186/1477-5956-3-6

中图分类号：

学科分类号：

摘要：

Background: The Observed molecular weight of a protein on a 1D polyacrylamide gel can provide meaningful insight into its biological function. Differences between a protein's observed molecular weight and that predicted by its full length amino acid sequence can be the result of different types of post-translational events, such as alternative splicing (AS), endoproteolytic processing (EPP), and post-translational modifications (PTMs). The characterization of these events is one of the important goals of total proteome profiling (TPP). LC/MS/MS has emerged as one of the primary tools for TPP, but since this method identifies tryptic fragments of proteins, it has not generally been used for large-scale determination of the molecular weight of intact proteins in complex mixtures. Results: We have developed a set of computational tools for extracting molecular weight information of intact proteins from total proteome profiles in a high throughput manner using 1D-PAGE and LC/MS/MS. We have applied this technology to the proteome profile of a human lymphoblastoid cell line under standard culture conditions. From a total of 1 x 107 cells, we identified 821 proteins by at least two tryptic peptides. Additionally, these 821 proteins are well-localized on the 1D-SDS gel. 656 proteins (80%) occur in gel slices in which the observed molecular weight of the protein is consistent with its predicted full-length sequence. A total of 165 proteins (20%) are observed to have molecular weights that differ from their predicted full-length sequence. We explore these molecular-weightdifferences based on existing protein annotation. Conclusions: We demonstrate that the determination of intact protein molecular weight can be achieved in a high-throughput manner using 1D-PAGE and LC/MS/MS. The ability to determine the molecular weight of intact proteins represents a further step in our ability to characterize gene expression at the protein level. The identification of 165 proteins whose observed molecular weight differs from the molecular weight of the predicted full-length sequence provides another entry point into the high-throughput characterization of protein modification. © 2005 Ahmad et al., licensee BioMed Central Ltd.

引用

页数：14

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