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 beta, HINT and alpha-enolase. Sequence modifications or conflicts were observed for beta-and gamma-actin, ATP beta-synthase and heat shock 90 beta. 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.

Development of new methodologies for the mass spectrometry study of bioorganic macromolecules

“…With suitable choices of those elements, mass spectrometry has become a very powerful tool for the analysis of bioorganic macromolecules mainly due to the development of new ionization methods; i.e., fast atom bombardment (FAB) Morris et al, 1981;Nichols & McMeekin, 2002), electrospray ionization (ESI) (Whitehouse et al, 1985), and matrix-assisted laser desorption ionization (MALDI) (Karas & Hillekamp, 1988). Those methods have been widely described in the literature (Li et al, 1999;Basile et al, 2001;Cunsolo et al, 2001;Cozzolino et al, 2001;Fierens et al, 2001a,b;Madonna et al, 2001;Ogorzalek Loo et al, 2001;Wall et al, 2001), and their mechanisms have been amply discussed; thus, we report here only on the main points of their analytical power.…”

Protein microarrays using liquid phase fractionation of cell lysates

“…Thus, the sensitivity and specificity for mitochondrial creatine kinase was 83%. Interestingly, mitochondrial creatine kinase has been associated with adenocarcinoma and its detection in sera has been correlated to poor outcome of the cancer [39]. In this regard our results showing the presence of auto-antibody repertoire could be due to overexpression of this protein in PCA.…”

Transforming omics data into context: Bioinformatics on genomics and proteomics raw data

“…Technological refinements have been realized as surfaceenhanced laser desorption/ionization-TOF-MS (SELDI-TOF-MS) [18], LC-MS [19], and Fourier transform ion cyclotron resonance-MS (FTICR-MS).…”