Improved Proteome Coverage by Using High Efficiency Cysteinyl-peptide Enrichment: The Human Mammary Epithelial Cell Proteome
Automated multidimensional capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly applied in various large scale proteome profiling efforts. However, comprehensive global proteome analysis remains technically challenging due to issues associated with sample complexity and dynamic range of protein abundances, which is particularly apparent in mammalian biological systems. We report here the application of a high efficiency cysteinyl-peptide enrichment (CPE) approach to the global proteome analysis of human mammary epithelial cells (HMECs) which significantly improved both sequence coverage of protein identifications and the overall proteome coverage. The cysteinyl-peptides were specifically enriched by using a thiol-specific covalent resin, fractionated by strong cation exchange chromatography, and subsequently analyzed by reversed-phase capillary LC-MS/MS. An HMEC tryptic digest without CPE was also fractionated and analyzed under the same conditions for comparison. The combined analyses of HMEC tryptic digests with and without CPE resulted in a total of 14,416 confidently identified peptides covering 4,294 different proteins with an estimated 10% gene coverage of the human geome. By using the high efficiency CPE, an additional 1,096 relatively low abundance proteins were identified, resulting in 34.3% increase in proteome coverage; 1,390 proteomes were observed with increased sequence coverage. Comparative protein distribution analyses revealed that the CPE method is not biased by protein molecular weight, pI, gene location, cellular location, or biological functions. These results demonstrate that the use of the CPE approach provides improved efficiency in comprehensive proteome-wide analyses of highly complex mammalian biological systems.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
- Sponsoring Organization:
- US Department of Energy (US)
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 15016081
- Report Number(s):
- PNNL-SA-42468; 10198; 3056; 400412000; TRN: US200509%%909
- Journal Information:
- Proteomics, Vol. 5, Issue 5; Other Information: PBD: 5 Apr 2005
- Country of Publication:
- United States
- Language:
- English
Similar Records
Characterization of the Mouse Brain Proteome Using Global Proteomic Analysis Complemented with Cysteinyl-Peptide Enrichment
High throughput comparative proteome analysis using a quantitative cysteinyl-peptide enrichment technology