Comprehensive analysis of proteins of pH fractionated samples using monolithic LC/MS/MS, intact MW measurement and MALDI-QIT-TOF MS.
A comprehensive platform that integrates information from the protein and peptide levels by combining various MS techniques has been employed for the analysis of proteins in fully malignant human breast cancer cells. The cell lysates were subjected to chromatofocusing fractionation, followed by tryptic digestion of pH fractions for on-line monolithic RP-HPLC interfaced with linear ion trap MS analysis for rapid protein identification. This unique approach of direct analysis of pH fractions resulted in the identification of large numbers of proteins from several selected pH fractions, in which approximately 1.5 microg of each of the pH fraction digests was consumed for an analysis time of ca 50 min. In order to combine valuable information retained at the protein level with the protein identifications obtained from the peptide level information, the same pH fraction was analyzed using nonporous (NPS)-RP-HPLC/ESI-TOF MS to obtain intact protein MW measurements. In order to further validate the protein identification procedures from the fraction digest analysis, NPS-RP-HPLC separation was performed for off-line protein collection to closely examine each protein using MALDI-TOF MS and MALDI-quadrupole ion trap (QIT)-TOF MS, and excellent agreement of protein identifications was consistently observed. It was also observed that the comparison to intact MW and other MS information was particularly useful for analyzing proteins whose identifications were suggested by one sequenced peptide from fraction digest analysis.
Yoo
Chul
C
Department of Chemistry, The University of Michigan, Ann Arbor, MI 48109, USA.
Patwa
Tasneem H
TH
Kreunin
Paweena
P
Miller
Fred R
FR
Huber
Christian G
CG
Nesvizhskii
Alexey I
AI
Lubman
David M
DM
D000595:Amino Acid Sequence; D014408:Biomarkers, Tumor / Q000032:analysis / Q000737:chemistry*; D001943:Breast Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000032:analysis / Q000737:chemistry*; D010449:Peptide Mapping / Q000379:methods*; D020539:Sequence Analysis, Protein / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D014408:Biomarkers, Tumor; D009363:Neoplasm Proteins