High Throughput Profiling of Serum Phosphoproteins/Peptides Using the SELDI-TOF-MS Platform

Abstract

Protein phosphorylation is a dynamic post-translational modification that plays a critical role in the regulation of a wide spectrum of biological events and cellular functions including signal transduction, gene expression, cell proliferation, and apoptosis. Determination of the sites and magnitudes of protein phosphorylation has been an essential step in the analysis of the control of many biological systems. A high throughput analysis of phosphorylation of proteins would provide a simple, logical, and useful tool for a functional dissection and prediction of biological functions and signaling pathways in association with these important molecular events. We have developed a functional proteomics technique using the ProteinChip array-based SELDI-TOF-MS analysis for high throughput profiling of phosphoproteins/phosphopeptides in human serum for the early detection and diagnosis as well as for the molecular staging of human cancer. The methodology and experimental approach consists of five steps: (1) generation of a total peptide pool of serum proteins by a global trypsin digestion; (2) rapid isolation of phosphopeptides from the total serum peptide pool by an affinity selection, purification, and enrichment using a novel automated micro-bioprocessing system with phospho-antibody-conjugated paramagnetic beads and a hybrid magnet plate; (3) high throughput phosphopeptide analysis on ProteinChip arrays by automated SELDI-TOF-MS; and (4) bioinformatics and statistical methods for data analysis. This method with appropriate modifications may be equally applicable to serine-, threonine- and tyrosine-phosphorylated proteins and for selectively isolating, profiling, and identifying phosphopeptides present in a highly complex phosphor-peptide mixture prepared from various human specimens such as cells, tissue samples, and serum and other body fluids.

CoFe2O4–ZnO nanoparticles for rapid microwave‐assisted tryptic digestion of phosphoprotein and phosphopeptide analysis by matrix‐assisted laser desorption/ionization mass spectrometry

“…Human blood serum is a complex system which contains abundant salt ions, proteins, phosphoproteins, and phosphopeptides. However, the separation of phosphoproteins or phosphopeptides from the serum is a challenging task . The phosphorylated fibrinopeptide A molecule is a well‐known catalytically efficient enzyme complex that is required for regulation of the blood coagulation system .…”

“…However, the separation of phosphoproteins or phosphopeptides from the serum is a challenging task . The phosphorylated fibrinopeptide A molecule is a well‐known catalytically efficient enzyme complex that is required for regulation of the blood coagulation system . Fibrinopeptides are released in blood with enzymatic reaction that leads to exposure of fibrin polymerization sites forming a resilient insoluble blood clot at the site of a wound.…”

“…[42] The phosphorylated fibrinopeptide A molecule is a well-known catalytically efficient enzyme complex that is required for regulation of the blood coagulation system. [42,43] Fibrinopeptides are released in blood with enzymatic reaction that leads to exposure of fibrin polymerization sites forming a resilient insoluble blood clot at the site of a wound. CoFe 2 O 4 -ZnO NPs were used to check the selectivity and affinity for human serum phosphopeptides.…”

Silica–Lanthanum Oxide: Pioneer Composite of Rare-Earth Metal Oxide in Selective Phosphopeptides Enrichment

“…However, direct determination of these modified proteins in a biological system has been difficult, because of the serum complexity and lower concentrations. There is no method available to study this aspect in detail with consistency . In order to estimate the detection limit for phosphopeptides enriched from serum, a sensitivity assessment is done using spiked serum with tryptic digest of β-casein in different ratios (40 μL serum sample spiked with 1 μg (50 pmol), 100 ng (5 pmol), 10 ng (500 fmol), and/or 1 ng (50 fmol) β-casein).…”

“…There is no method available to study this aspect in detail with consistency. 42 In order to estimate the detection limit for phosphopeptides enriched from serum, a sensitivity assessment is done using spiked serum with tryptic digest of β-casein in different ratios (40 μL serum sample spiked with 1 μg (50 pmol), 100 ng (5 pmol), 10 ng (500 fmol), and/or 1 ng (50 fmol) β-casein). The identified phosphopeptides from β-casein are at m/z 1103 (KFQS*EEQQQT), 1473 (KKIEKFQS*EEQQQT), and 2061 (FQS*EEQQQTEDELQDK), with relatively good MS peak intensity.…”