期刊
PROTEOMICS
卷 7, 期 2, 页码 299-312出版社
WILEY
DOI: 10.1002/pmic.200600272
关键词
cancer biomarkers; electrospray ionization time-of-flight mass spectrometry; liquid chromatography; metastasis; monolith
资金
- NATIONAL CANCER INSTITUTE [R01CA108597] Funding Source: NIH RePORTER
- NCI NIH HHS [R01 CA108597-04, R01 CA108597] Funding Source: Medline
A combination of LC and MS was applied to an isogenic breast tumor metastasis model to identify proteins associated with a cellular phenotype. Chromatofocusing followed by nonporous-RP-HPLC/ESI-TOF MS was applied to cell lysates of a pair of monoclonal cell lines from the human breast carcinoma cell line MDA-MB-435 that have different metastatic phenotypes in immune-compromised mice. This method was developed to separate proteins based on pI and hydrophobicity. The high resolution and mass accuracy of ES1-TOF measurements provided a good correlation of theoretical MW and experimental M-r values of intact proteins measured in mass maps obtained in the pH range 3.8-6.4. The isolated proteins were digested by trypsin and analyzed by MALDI-TOF NIS, MALDI-QIT-TOF MS, and monolith-based HPLC/MS/MS. The unique combination of the techniques provided valuable information including quantitation and modification of proteins. We identified 89 selected proteins, of which 43 were confirmed as differentially expressed. Metastasis -associated proteins included galectin-1, whereas annexin I and annexin 11 were associated with the nonmetastatic phenotype. In this study, we demonstrate that combining a variety of MS tools with a multidimensional liquid-phase separation provides the ability to map cellular protein content, to search for modified proteins, and to correlate protein expression with cellular phenotype.
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