Phosphopeptide ions

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.

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... 

Li, S. and Dass, C., Iron(III)-Immobilized Metal Ion Affinity Chromatography and Mass Spectrometry for the Purification and Characterization of Synthetic Phosphopeptides, Anal. Biochem., 270, 9, 1999. 

Cao P. and Stults J.T. (1999), Phosphopeptide analysis by on-line immobilized metal-ion affinity chromatography-capillary electrophoresis-electrospray ionization mass spectrometry, J. Chromatogr. 853(1-2), 225-235. 

The synthesis of phosphopeptides is typically confirmed mass spectrometri-cally using either a MALDI (matrix-assisted laser desorption/ionizafion) or an ESI (electrospray ionization) source, and the peptide purity is determined by reversed-phase chromatography coupled to an UV detector (Figs. 1 and 2B). Whenever possible, phosphopeptide analyses should be complimented by mass spectra recorded in negative ion mode. For most biochemical applications it is necessary to purify the peptides by HPLC techniques (Fig. 2A). 

For details on MALDI or ESI mass spectrometry, see Chapter 3. For MALDI-MS the phosphopeptides are cocrystallized with pCHCA and measured in positive and negative ion mode (Fig. 3). Addition of phosphate to CHCA, which is commonly used for peptides, increases the signal intensities of phosphopeptides relative to unphosphorylated peptides. For multiphosphory-lated peptides we typically use DHB to further increase the signal intensity. However, these matrix effects depend on the instrument and cannot be generalized. 

If polar sequences or short phosphopeptides fail to bind to the stationary phase upon loading, the acetonitrile content of the eluent could be reduced to 3%. A further decrease is not possible for standard Ci8-columns, as the stationary phase could collapse reducing the loading capacity and the reproducibility. Instead, TFA could be replaced by the more hydrophobic ion-pair reagent HFBA (heptafluorobutyric acid), or a stationary phase with a special coating compatible with pure aqueous eluents could be used (e.g., Aqua-column from Phenomenex). 

Figure 19-15. LC/ESI-MS/MS product ion mass spectrum of phosphopeptides from Glu-C/chymotrypsin-digested MEKl.

H. Steen, B. Kuster, and M. Mann, Quadrupole time-of-flight versus triple-quadrupole mass spectrometry for the determination of phosphopeptides by precursor ion scanning, /. Mass Spectrom. 36 (2001), 782-790. 

In a study on gel filtration of tryptic hydrolyzates of a-casein, Bennich (1961) presented results of interest in this connection. On Sephadex G-25 he obtained a single peak containing bound hexoses. A gel as expanded as Sephadex G-75 was necessary to reveal the presence of several hexose-containing peptides. They are probably of different molecular size. The intermediate swelling grade of Sephadex, G-50, gave optimum conditions for separation of phosphopeptides. Because of their strongly acidic properties these peptides are probably subjected to some kind of ion exclusion... 

To address some of the issues associated with CID of phos-phopeptides, new strategies for ion activation/dissociation have been introduced recently in the context of phosphoproteomics. In particular, electron transfer dissociation (ETD) is emerging as a promising new strategy for MS/MS-based phosphopeptide analysis (22, 23). 

It should also be noted that besides the conventional production scanning, specialized MS/MS functions have been adopted for phosphopeptide analysis (9). These include precursor ion scanning for monitoring the precursors of the product ion POs" m/z 79) in the negative mode, or precursors of the phosphotyrosine-specihc immonium ion at m/z 216.043 in the positive mode. 

Niihse T, Yu K, Salomon A. Isolation of phosphopeptides by immobilized metal ion affinity chromatography. Curr. Protoc. Mol. Biol. 2007 18 18.13. 

Figure 4. Low-energy collision tandem MS of the (M+2H) (m/z 748) for the early-eluting phosphopeptide in Fig. 3. The asterisks indicate phosphate losses from the next higher mass bn-ion. The pS indicates the phosphorylated serine.

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