Reference-facilitated Phosphoproteomics

Abstract

Recent advances in instrument control and enrichment procedures have enabled us to quantify large numbers of phosphoproteins and record site-specific phosphorylation events. An intriguing problem that has arisen with these advances is to accurately validate where phosphorylation events occur, if possible, in an automated manner. The problem is difficult because MS/MS spectra of phosphopeptides are generally more complicated than those of unmodified peptides. For large scale studies, the problem is even more evident because phosphorylation sites are based on single peptide identifications in contrast to protein identifications where at least two peptides from the same protein are required for identification. To address this problem we have developed an integrated strategy that increases the reliability and ease for phosphopeptide validation. We have developed an off-line titanium dioxide (TiO 2 ) selective phosphopeptide enrichment procedure for crude cell lysates. Following enrichment, half of the phosphopeptide fractionated sample is enzymatically dephosphorylated, after which both samples are subjected to LC-MS/ MS. From the resulting MS/MS analyses, the dephosphorylated peptide is used as a reference spectrum against the original phosphopeptide spectrum, in effect generating two peptide spectra for the same amino acid sequence, thereby enhancing the probability of a correct identification. The integrated procedure is summarized as follows: 1) enrichment for phosphopeptides by TiO 2 chromatography, 2) dephosphorylation of half the sample, 3) LC-MS/MS-based analysis of phosphopeptides and corresponding dephosphorylated peptides, 4) comparison of peptide elution profiles before and after dephosphorylation to confirm phosphorylation, and 5) comparison of MS/MS spectra before and after dephosphorylation to validate the phosphopeptide and its phosphorylation site. This phosphopeptide identification represents a major improvement as compared with identifications based only on single MS/MS spectra and probability-based database searches. We investigated an applicability of this method to crude cell ly- Reversible protein phosphorylation, at specific serine, threonine, and tyrosine residues, is a key determinant in many fundamental cellular functions such as survival, differentiation, structural organization, and stress responses (1-4). Significant progress has been made in phosphopeptide identification at the femtomole level as phosphoproteomics, which permits rapid and effective identification and quantification of a large number of phosphoproteins (5-18). Large scale phosphoproteomics analysis generally depends on selective proteolytic digestions, selective phosphopeptide enrichment methods, and sensitive and informative LC-MS/MS. However, there are several unsolved issues regarding the use of these techniques in biological applications. Although recent improvements of phosphopeptide enrichment methods have raised the expectations for the field of phosphoproteomics (5, 9, 17, 19 -21), the technologies used are stil...

In Vivo Identification of Sumoylation Sites by a Signature Tag and Cysteine-targeted Affinity Purification

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Immobilized metal affinity chromatography using open tubular capillary for phosphoprotein analysis: Comparison between polymer brush coating and surface functionalization

“…The generated peaks are detailed in Table 1. [35,37,51,52] using excess phosphate and acetonitrile hydrophobicity to release the peptides. Both strongly and slightly basic conditions (Fig.…”

Understanding cell signaling in cancer stem cells for targeted therapy – can phosphoproteomics help to reveal the secrets?

“…This first dimension is usually combined with a final (ion-pair) reversed-phase (IP-RP) separation before mass spectrometric detection via high-resolution mass spectrometry (HRMS) [51]. Offering the advantage of very high resolution and mass accuracy, high-resolution hybrid mass spectrometers such as quadrupole-time-of-flight (Q-TOF) [75], linear ion trap-Orbitrap (LTQ-Orbitrap), or quadrupole-Orbitrap (Q-Orbitrap) instruments [76] are the first choice in large-scale phosphoproteomics approaches. These instruments provide full scan spectra of intact peptides as well as fragment spectra of selected peptide precursor ions, which are then compared with databases for peptide identification by means of suitable computational tools [55, 56, 77].…”