Phosphorylated peptides, selective detection

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. 

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... 

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.)...

Thus, a variety of mass spectrometry techniques are available that can selectively detect phosphoproteins and peptides in complex samples and can provide the site of phosphorylation in a protein. 

In order to enhance affinity and selectivity for Brc-Abl, we modified the inhibitor methylating at positions I and II (Fig. 7.5d). The synthesis of the wrapping prototype recapitulates imatinib synthesis [38], as described in [39], To test whether the specificity and affinity for Brc-Abl improved, we conducted a spectrophotometric kinetic assay to measure the phosphorylation rate of peptide substrates in the presence of the kinase inhibitor at different concentrations. This assay couples production of adenosine diphosphate (ADP), the byproduct of downstream phosphorylation, with the concurrent detectable oxidation of reduced nicotinamide adenosine dinucleotide (NADH). The oxidation results upon transfer of phosphate from PEP (phospho-enolpyruvate) to ADP followed by the NADH-mediated reduction of PEP to lactate. Thus, phosphorylation activity is monitored by the decrease in 340 nm absorbance due to the oxidative conversion NADH->-NAD+ [34, 39]. 

Detection of phosphorylated proteins and peptides can be enhanced by selectively isolating these species. Online immobilized metal affinity chromatography (IMAC)-CE-ESI-MS is such a powerful analytical tool. The IMAC resin retains and preconcentrates phosphorylated proteins and peptides, CE separates the phosphorylated species and MS/MS identifies the components and their phosphorylation sites. Cao and Stults applied this method to the analysis of phosphorylated angiotensin II and tryptic digests of a- and /3-casein (CE conditions buffer, 0.1% acetic acid/10% methanol uncoated capillary). Beta-casein is a well-characterized protein with five phosphorylation sites and is widely used as a standard for protein phosphorylation studies. 

Although an LC/MS combination provides high-resolution separation and the specific detection of analytes in a complex mixture, in many situations it is necessary to provide purification, concentration, and isolation of desired components (e.g., in a biological matrix). This step is performed by affinity chromatography. Practical examples are the selective capture of a specific antigen by the corresponding antibody and the isolation of phosphorylated proteins and peptides [96,97]. An... 

Most compounds treated so far are suitable for the derivatization of amino groups in peptides and amino acids. In addition, selective labels for the sulfhydryl group of cysteine have been reported [202-204]. For the labeling of gluthathione 80, both ferrocenyl-ethylmaleimide 81 and ferrocenyl-iodoacetamide 82 (Scheme 5.42) turned out to be very effective to yield 83 and 84, respectively [202, 204]. A related maleimide derivative has also been used to label the hexapeptide Ac-Arg-Arg-Ala-Ser-Leu-Cys-OH. This labeled hexapeptide was applied for the detection of serine phosphorylation by protein kinase A via electrochemical methods [203]. 

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