Proteome profile of human breast cancer tissue generated by LC-ESI-MS/MS combined with sequential protein precipitation and solubilization.
Comprehensive proteome profiling of breast cancer tissue samples is challenging, as the tissue samples contain many proteins with varying concentrations and modifications. We report an effective sample preparation strategy combined with liquid chromatography (LC) electrospray ionization (ESI) quadrupole time-of-flight (QTOF) MS/MS for proteome analysis of human breast cancer tissue. The complexity of the breast cancer tissue proteome was reduced by using protein precipitation from a tissue extract, followed by sequential protein solubilization in solvents of different solubilizing strength. The individual fractions of protein mixtures or subproteomes were subjected to trypsin digestion and the resultant peptides were separated by strong cation exchange (SCX) chromatography, followed by reversed-phase capillary LC combined with high resolution and high accuracy ESI-QTOF MS/MS. This approach identified 14407 unique peptides from 3749 different proteins based on peptide matches with scores above the threshold scores at the 95% confidence level in MASCOT database search of the acquired MS/MS spectra. The false positive rate of peptide matches was determined to be 0.95% by using the target-decoy sequence search strategy. On the basis of gene ontology categorization, the identified proteins represented a wide variety of biological functions, cellular processes, and cellular locations.
Gong
Yan
Y
PolyomX Program, Cross Cancer Institute, Alberta Cancer Board, and Department of Chemistry, Oncology, and Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada.
Wang
Nan
N
Wu
Fang
F
Cass
Carol E
CE
Damaraju
Sambasivarao
S
Mackey
John R
JR
Li
Liang
L
D019840:2-Propanol; D000096:Acetone; D001943:Breast Neoplasms / Q000378:metabolism*; D005591:Chemical Fractionation; D011232:Chemical Precipitation; D002853:Chromatography, Liquid; D005260:Female; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000302:isolation & purification; D012997:Solvents; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D008565:Membrane Proteins; D020543:Proteome; D012997:Solvents; D000096:Acetone; D012313:RNA; D019840:2-Propanol