Isoelectric focusing nonporous silica reversed‐phase high‐performance liquid chromatography/electrospray ionization time‐of‐flight mass spectrometry: a three‐dimensional liquid‐phase protein separation method as applied to the human erythroleukemia cell‐line

Abstract

A liquid‐phase three‐dimensional protein separation method has been developed that is used to separate the cytosolic fraction of a HEL cell lysate via isoelectric focusing (IEF), nonporous silica (NPS) reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and electrospray ionization time‐of‐flight mass spectrometry (ESI‐TOFMS), respectively. Several hundred unique protein molecular weights were observed in a pI range from 4.8 to 8.5 and a mass range from 5 to 85 kDa. Proteins were positively identified by analysis of the pI (±0.5 pI units), an intact protein molecular weight (±150 ppm), and peptide mass mapping results. Using the molecular weight (MW) and peptide mapping results of identified proteins it was possible to characterize their posttranslational (PTMs) and/or sequence modifications. PTMs were detected on both forms of cytosolic actin, heat shock 90 β, HINT and α‐enolase. Sequence modifications or conflicts were observed for β‐and γ‐actin, ATP β‐synthase and heat shock 90 β. IEF‐NPS‐RP‐HPLC/ESI‐TOFMS was used to determine experimental pI, MW and relative hydrophobicity values for each protein detected. This data was used to generate a 2‐D pI‐MS protein map, where proteins are displayed according to their pI and molecular weight. Protein molecular weight peaks are represented as bands in the 2‐D pI‐MS image where the gray scale of each band is proportional to the intensity of the protein molecular weight peak. In addition, a third hydrophobicity dimension (%B) was added as the % acetonitrile elution to generate a 3‐D pI‐MS‐%B plot where each protein can be tagged according to three parameters. Copyright © 2001 John Wiley & Sons, Ltd.