Monolithic column HPLC separation of intact proteins analyzed by LC-MALDI using on-plate digestion: An approach to integrate protein separation and identification.
A method is developed to integrate a protein separation by monolithic capillary reversed-phase high-performance liquid chromatography to on-probe tryptic digestion for subsequent analyses by MALDI-TOF MS and MALDI-TOF/TOF MS. The method provides a means of directly interfacing separations to MALDI-MS, reducing the amount of time required for traditional procedures involving in-solution enzymatic digestion and sample cleanup prior to MALDI-MS analysis. When used with pI-based fractionation as a first dimension, it provides a means of analyzing complex mixtures of proteins with minimal sample handling and cleanup. The use of monolithic capillary columns sufficiently resolved intact proteins so that peptide mass fingerprinting analysis by MALDI-TOF MS resulted in the identification of close to 40 unique proteins from 120 ng of sample obtained from a prefractionated MCF10 cell line at pH 6.34, where the identifications of several of these proteins were also confirmed by intact MW and tandem mass spectrometric analysis. The reproducibility of this method has been demonstrated to be sufficient for the purpose of protein identifications. Experimental values of protein intact MW are obtained and compared to that expected for each protein identified.
Zheng
Suping
S
Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109, USA.
Yoo
Chul
C
Delmotte
Nathanael
N
Miller
Fred R
FR
Huber
Christian G
CG
Lubman
David M
DM
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation* / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009862:Online Systems; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation* / Q000379:methods*
D011506:Proteins