title: 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.
abstract: 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.
authors: Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA.
authors: Dipak
authors: D
authors: Thakur
authors: Tomas
authors: T
authors: Rejtar
authors: Dongdong
authors: D
authors: Wang
authors: Jonathan
authors: J
authors: Bones
authors: Sangwon
authors: S
authors: Cha
authors: Buffie
authors: B
authors: Clodfelder-Miller
authors: Elizabeth
authors: E
authors: Richardson
authors: Shemeica
authors: S
authors: Binns
authors: Sonika
authors: S
authors: Dahiya
authors: Dennis
authors: D
authors: Sgroi
authors: Barry L
authors: BL
authors: Karger
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods*; D016000:Cluster Analysis; D003584:Cytological Techniques / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005260:Female; D022781:Hepatocytes; D006801:Humans; D061346:Laser Capture Microdissection; D008198:Lymph Nodes; D051379:Mice; D010446:Peptide Fragments; D016062:Porosity; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000145:classification; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D010446:Peptide Fragments; D011506:Proteins