Microproteomic analysis of 10,000 laser captured microdissected breast tumor cells using short-range sodium dodecyl sulfate-polyacrylamide gel electrophoresis and porous layer open tubular liquid chromatography tandem mass spectrometry

• Published in: Journal of Chromatography A 2011-11, Vol.1218 (45), p.8168-8174
• Main Authors: Thakur, Dipak, Rejtar, Tomas, Wang, Dongdong, Bones, Jonathan, Cha, Sangwon, Clodfelder-Miller, Buffie, Richardson, Elizabeth, Binns, Shemeica, Dahiya, Sonika, Sgroi, Dennis, Karger, Barry L.
• Format: Article
• Language: English
• Subjects: Breast cancer; breast neoplasms; fractionation; hepatocytes; Laser capture microdissection; liquid chromatography; Low cell numbers; metastasis; mice; Microproteomics; neoplasm cells; patients; peptides; polyacrylamide gel electrophoresis; Porous layer open tubular column; protein synthesis; proteins; proteomics; Sample preparation; tandem mass spectrometry
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2011.09.022

► A microproteomic method for limited LCM collected cells is presented. ► Method comprises short range SDS-PAGE and ultrasensitive PLOT LC-MS. ► Over 1100 proteins routinely identified from a digest corresponding to only 1000 cells. ► Relevant and informative biological meaning was extracted from this limited cell analysis. Precise proteomic profiling of limited levels of disease tissue represents an extremely challenging task. Here, we present an effective and reproducible microproteomic workflow for sample sizes of only 10,000 cells that integrates selective sample procurement via laser capture microdissection (LCM), sample clean-up and protein level fractionation using short-range SDS-PAGE, followed by ultrasensitive LC–MS/MS analysis using a 10 μm i.d. porous layer open tubular (PLOT) column. With 10,000 LCM captured mouse hepatocytes for method development and performance assessment, only 10% of the in-gel digest, equivalent to ∼1000 cells, was needed per LC–MS/MS analysis. The optimized workflow was applied to the differential proteomic analysis of 10,000 LCM collected primary and metastatic breast cancer cells from the same patient. More than 1100 proteins were identified from each injection with >1700 proteins identified from three LCM samples of 10,000 cells from the same patient (1123 with at least two unique peptides). Label free quantitation (spectral counting) was performed to identify differential protein expression between the primary and metastatic cell populations. Informatics analysis of the resulting data indicated that vesicular transport and extracellular remodeling processes were significantly altered between the two cell types. The ability to extract meaningful biological information from limited, but highly informative cell populations demonstrates the significant benefits of the described microproteomic workflow.