Phosphorylated peptides, detection

A more traditional but still successful method for the detection of a protein phosphorylation is by radioactive labeling with 35P. The labeled protein is digested, the peptides are separated by high-performance liquid chromatography, and the phosphorylated peptides are detected in specific fractions via their radioactivity. The fraction with the phosphorylated peptides can be further analyzed by mass spectrometry (Figeys et al., 1999). 

Fig. 9. Precursor ion scan on an electrospray triple quadrupole mass spectrometer. From all the peptides present of the digested protein only those that are phosphorylated are detected in a precursor ion scan for the phosphate ion (P03, mass 79 Da) in negative ion mode. From the TPX protein three phosphorylated peptides could be detected Ml, AQLTM PSTPTVLK M2, LSETSVNTEQNSK and M3, VQPVQTTPSKDDVSNSATHVC DVK. M, Oxidized methionine C, carbamidomethylated cysteine.

Inamori, K., Kyo, M., Nishiya, Y, Inoue, Y., Sonoda, T., Kinoshita, E., Koike, T. and Katayama, Y. (2005) Detection and quantification of on-chip phosphorylated peptides by surface plasmon resonance imaging techniques... 

Nakanishi T, Ando E, Furuta M, Tsunasawa S, Nishimura O (2007) Direct on-membrane peptide mass fingerprinting with MALDI-MS of tyrosine-phosphorylated protein detected by immunostaining. J Chromatogr B 847 24-29. doi 10.1016/j.jchromb.2006.08.024... 

Our initial applications of the stepped collision energy LC-ESMS approach involved selective detection (and differentiation) of N- or O-glycosylated peptides (5,6), and detection of phosphorylated peptides (7,8,11). The method has been in routine use in our laboratory for over two years and during this time has become one of the mainstays of our work in characterizing protein modifications. Here we present some of our more recent studies on protein glycosylation and phosphorylation, and illustrate a preliminary evaluation of the stepped collision energy LC-ESMS method for selective detection of sulfated and acylated peptides in protein digests. 

H. Steen, B. Ktlster, M. Fernandez, A. Pandey, M. Mann, Detection of Tyr phosphorylated peptides by precursor ion scanning Q-TOF-MS in positive-ion mode. Anal. Chem., 73 (2001) 1440. 

J.G. Krabbe, H. Lingeman, W.M.A. Niessen, H. Irth, Ligand-exchange detection of phosphorylated peptides using LC-ESI-MS, Anal. Chem., 75 (2003) 6853. 

In vitro colorimetric assays were performed over a 1 pM to 100 (xM range in ligand concentration to assess the inhibition of phosphorylating activity by antibody recognition of phosphorylated peptide substrates [14]. The IC50 (50% inhibition concentration) for imatinib/Abl is 1 (xM, while the WBZ 4/Abl value is above 100 (xM (Fig. 8.7a). The active recombinant Abl kinase and its substrate (Abl-tide) were incubated in the presence of various WBZ 4 or imatinib concentrations and ATP (100 nM). Phosphorylation of Abl-tide peptide was detected by spectrophotometry following incubation with phospho-Abl-tide antibodies. 

Under CID, all three types of O-phosphorylated peptides exhibit the loss of 98 Da due to the expulsion of H3PO4 (and/or HP03 + H20). This reaction has been exploited to detect phosphopeptides in a protein digest with neutral-loss scan.103... 

Fig. 16 uses the implementation of a tyrosine kinase assay as an example to illustrate the two major assay formats. The task of the assay is to detect the kinase activity, i. e., the amount of phosphorylated peptide that is produced during the enzymatic reaction. 

The significant emission enhancement of these chemosensors and the high selectivity towards phosphorylated peptides enabled the detection of phosphorylated peptides by naked inspection of the emission change. This is illustrated in the photograph shown in Fig. 5. Such fluorescence intensification of the chemosensors is clearly ascribed to the phosphate-assisted binding of the second Zn cation. A schematic illustration of the sensing mechanism toward the phosphorylated peptide is depicted in Scheme 11. In the absence of a phosphorylated peptide, the second Dpa site of the chemosensor is... 

Fig. 10.10 ESI-FTICR mass spectrum of tryptic digest mixture of multi- phosphorylated human neurofibrillary tau protein. Peak assignments within the m/z detection range, 200—2500, are shown for phosphorylated peptides. The insert shows the peptide fragment of the phosphorylation domain (512-538) the seven phos-...

APCE has also been used to investigate the fast kinetics of peptide-protein interactions. A fluorescently labeled phosphorylated peptide was mixed with a Src homology 2-domain protein and separations of complex and free probe could be achieved in less than 4 s. These rapid separations were necessary, because no complex peak was observed with 8 s separations, presumably because it had dissociated. Estimated dissociation constants for the complex were obtained from peak areas and results were similar to constants from fluorescence anisotropy data. Aptamers have also been used as affinity ligands for fast separations. Separations as short as 30 s were accomplished with a fluorescently labeled aptamer against IgE. " APCE has also been used to detect nM concentrations of digoxin in less than 1 min. ... 

Mass spectrometry of phosphopeptides has become a powerful tool for phosphorylation site identification. However, proteolytic digests examined by MS are often likely to fail to detect phosphopeptides because the ionization of phosphorylated peptides in positive ion mode MS is generally less efficient compared with the ionization of their nonphosphorylated counterparts resulting in ion suppression effects. A further problem is that phosphopeptides may not be retained by RP chromatography because they are too small and/or hydrophilic to bind to the CIS stationary phase. Therefore, capillary electrophoresis coupled to MS is a powerful method to enhance the detection of phosphoproteins and phosphopeptides due to their efficient separation by CE. 

With MALDI-MS detection, the microfluidic compact disk was demonstrated for the high-throughput analysis of protein digests and phosphorylated peptides.For protein digests. 

Figure 4.4. Precursor-ion scan of miz 79 for selective detection of phosphopeptides. Two tyrosine phosphorylated peptides (1303.2 and 1686.7 Da) are detected in an LC fraction. (Reproduced from ref. 5 by permission of the American Chemical Society, Washington, DC, copyright 1993.)...

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