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. 2009 Dec;8(12):5523-31.
doi: 10.1021/pr900603n.

An initial characterization of the serum phosphoproteome

Weidong Zhou  1 , Mark M Ross, Alessandra Tessitore, David Ornstein, Amy Vanmeter, Lance A Liotta, Emanuel F Petricoin 3rd
Affiliations

An initial characterization of the serum phosphoproteome

Weidong Zhou et al. J Proteome Res. 2009 Dec.

Abstract

Phosphorylation is a dynamic post-translational protein modification that is the basis of a general mechanism for maintaining and regulating protein structure and function, and of course underpins key cellular processes through signal transduction. In the last several years, many studies of large-scale profiling of phosphoproteins and mapping phosphorylation sites from cultured human cells or tissues by mass spectrometry technique have been published; however, there is little information on general (or global) phosphoproteomic characterization and description of the content of phosphoprotein analytes within the circulation. Circulating phosphoproteins and phosphopeptides could represent important disease biomarkers because of their well-known importance in cellular function, and these analytes frequently are mutated and activated in human diseases such as cancer. Here, we report an initial attempt to characterize the phosphoprotein content of serum. To accomplish this, we developed a method in which phosphopeptides are enriched from digested serum proteins and analyzed by LC-MS/MS using LTQ-Orbitrap (CID) and LTQ-ETD mass spectrometers. With this approach, we identified approximately 100 unique phosphopeptides with stringent filtering criteria and a lower than 1% false discovery rate.

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Figures

Figure 1
Figure 1
Flowchart of the experimental procedure. Groups of serum samples were digested by trypsin or LysC, and phosphopeptides were purified by single/dual TiO2-enrichment strategies as well as immunoaffinity for LC-MS/MS analyses using LTQ-Orbitrap and/or LTQ-ETD, followed by phosphopeptide identification by database search and manual data evaluation.
Figure 2
Figure 2
Verification of identified phosphopeptide ETTCSKEpSNEELTESCETK from kininogen by MS2 spectra comparison. (a) CID spectrum of the phosphopeptide (2+ ion m/z 1171.4567) obtained from TiO2 enriched serum phosphopeptides; (b) CID spectrum of synthetic phosphopeptide ETTCSKEpSNEELTESCETK.
Figure 3
Figure 3
Example CID and ETD spectra of the doubly-charged phosphopeptide pSKEQLTPLIK from Apolipoprotein A-II. (a) CID spectrum labeled to show singly-charged b and y ions, as well as ions corresponding to neutral losses of the phosphate group and water. (b) ETD spectrum labeled to show singly-charged c and z ions, as well as ions corresponding to unfragmented and charge-reduced parent ion.
See this image and copyright information in PMC

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