Analysis of phosphopeptides

Tholey, A., Ionic liquid matrices with phosphoric acid as matrix additive for the facilitated analysis of phosphopeptides by matrix-assisted laser desorption/ ionization mass spectrometry. Rapid Commun. Mass Spec., 20,1761, 2006. 

W. J. Qian, M. B. Goshe, D. G. Camp II, L.-R. Yu, K. Tang, and R. D. Smith, Phos-phoprotein isotope-coded solid-phase tag approach for enrichment and quantitative analysis of phosphopeptides from complex mixtures, A aZ. Chem.15 (2003), 5441-5450. 

Kjellstrom, S. and Jensen, O.N. (2004) Phosphoric acid as a matrix additive for MALDI MS analysis of phosphopeptides andphosphoproteins. Anal. Chem. 76, 5109-5117. 

As mentioned above, main concerns in the mass spectrometry analysis of phosphopeptides in a complex protein digest are their low amounts and signal suppression in the presence of unphosphorylated peptides. To alleviate these concerns, the isolation and enrichment of phosphopeptides from the bulk of unphosphorylated peptides are highly desirable. A convenient means to fractionate phosphopeptides is RP-HPLC. Offline and online procedures with mass spectrometry have... 

H. Steen and M. Mann, A new derivatization strategy for the analysis of phosphopeptides by precursor ion scanning in positive ion mode, 7. Am. Soc. Mass Spectrom. 13, 996-1003 (2002). 

Chen, C.T. and Chen, Y.-C. (2005) Fe304/Ti02 core/shell nanoparticles as affinity probes for the analysis of phosphopeptides using Ti02 surface-assisted laser desorption/ionization mass spectrometry. Anal. Chem., 77, 5912-5919. 

It has been shown [18] that when high cone-voltages were used in conjunction with negative-ion electrospray, phosphopeptides produce diagnostic ions at m/z 63 (P02 ) and m/z 79 (P03 ). LC-MS analysis of a trypsin digest of bovine... 

Identification of associated protein kinases Based on phosphopeptide analyses it became clear that associated kinases modify principally serine and threonine residues. Moreover, the analysis of putative phosphorylation-speciflc consensus sequences of p53, c-Jun, p27, ICSBP and I/cBa revealed that the protein kinase CK2 and a member of the protein kinase C family might be associated with the CSN. 

Competition binding analysis of a variety of phosphopeptides derived from different Cdc4 substrates revealed a range of affinities. For example. Sic IP = 24 pM, FarlP = 2.7 pM, Gcn4 = 0.8 pM. Importantly, multi-site phosphorylation dependent interactions with Cdc4 can be recapitulated with synthetic concatamers of low affinity CPD sites based on either the regions T45 or S76 sites in Sicl. 

Dunkley PR, Robinson PJ (1986) Depolarization-dependent protein phosphorylation in synapto-somes mechanisms and significance. Prog Brain Res 69 273-93 Dunkley PR, Baker CM, Robinson PJ (1986) Depolarization-dependent protein phosphorylation in rat cortical synaptosomes characterization of active protein kinases by phosphopeptide analysis of substrates. J Neurochem 46 1692-1703... 

FIGURE 7.44 MALDI mass spectra from on-CD analysis of the phosphopeptide-con-taining sample, (a) Peptide mass spectrum after concentration/desalting. A database search showed that the sample contained bovine protein disulfide isomerase. (b) Phosphopeptide enrichment by IMAC. Two phosphopeptides at m/z 964 and 2027 were recognized (c) Phosphopeptide enrichment followed by enzymatic on-column dephosphorylation using alkaline phosphatase. Two phosphopeptides at m/z 884 and 1947 were recognized from the mass shifts of 80 Da from (B), which were resulted from dephosphorylation [794]. Reprinted with permission from the American Chemical Society. 

K20. Kuiper, G. G., and Brinkmann, A. O., Phosphotryptic peptide analysis of the human androgen receptor Detection of a hormone-induced phosphopeptide. Biochemistry 34,1851-1857 (1995). 

A comprehensive analysis of protein phosphorylation includes the identification of the phosphoprotein or phosphopeptide, the localization of the modified amino acid, and if possible, the quantification of phosphorylation. 

Figure 3 MS/MS spectrum of the phosphopeptide FNDS EGDDTEETEDYR. The spectrum, which was acquired with an ion trap mass spectrometer, illustrates the typical behavior of phosphopeptide ions under low-energy CID. The molecular ion was (M- -2EI)2+, m/z 1001.9. The MS/MS spectrum is dominated by an intense product ion corresponding to the neutral loss of phosphoric acid from the activated precursor ion. In addition, the spectrum contains a number of product ions from the y- and b-series that determine the peptide sequence and the site of phosphorylation. (The product ions are singly charged unless noted otherwise). This phosphopeptide belongs to Bcl-2-associated transcription factor 1 (BCEFI HUMAN) and it was identified in the analysis of the phosphoproteome in the LNCaP prostate cancer cell line.

The use of isotope-labelled synthetic peptides as IS was proposed for the absolute quantification of proteins in protein expression studies [113]. If necessary, these synthetic peptides can be covalently modified to be applied as IS in for instance phosphopeptide quantification. This approach was applied in the quantitative analysis of two glutathione 5 -transferase isoforms in human liver cytosol by LC-MS in SRM mode [114]. A series of pilot experiments were performed to select the most suitable IS peptides for four human plasma proteins (hemopexin, ocl antichymotrypsin, interleiddn-6, and tumor necrosis factor-oc) [115]. Rabbit polyclonal antibodies were raised against these selected peptides and immobilized on POROS supports. These lAC columns were applied to achieve a 120-fold emichment of the antigen peptide from digested human plasma proteins. The peptides and its IS were measured by LC-MS in SIM or SRM mode. The methods appears to be a tailor method for targeted protein analysis. 

While all these strategies rely on specific properties of phosphopeptides in MS analysis, a more global approach involving shotgun protein identification strategies and SEQUEST database searching (Ch. 18.3.2) is demonstrated by the group of Yates for protein complexes and lens tissue proteins [29]. 

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