Mass spectrometric characterization of phosphorylated muscle proteins

amino acid side chains such as serine, threonine, tyrosine or aspartic acid. The reverse reaction is called dephosphrylation. Protein phosphorylation changes the conformation, because the phosphate group is carrying two negative charges, which may act as a molecular switch, turning protein activity on and off. Both phosphorylation and dephosphorylation are catalysed by a wide variety of enzymes such as protein kinases and protein phophatases [183, 184]. Generally, the analysis of protein phosphorylation refers to phosphoproteomics, which involves identification of phosphoproteins and phosphopeptides and localization of the exact residues that are phosphorylated. However, the characterization of phosphoproteins is not simple for several reasons: generally the stoichiometry of phosphorylation is relatively low, therefore enrichment steps are a prerequisite prior analysis, most analytical techniques used for characterization of protein phosphorylation have a limited dynamic range and before analysis of phosphoproteins phosphatases activity must be inhibited, which otherwise, may dephosphorylate proteins during sample preparation step [185]. In a routine fashion may be used to immunoprecipitate specific tyrosine phosphorylated proteins enriching them from protein mixture such as cell lysate. In previous studies such antibodies were used successfully for enrichment of tyrosine phosphorylated proteins while they prove not to be so efficient to enrichment of phosphopeptides [186] and, therefore, alternative methods are needed. A possible alternative is immobilized metal affinity chromatography (IMAC), which exploits the hjgh affinity of phosphate groups towards a metal chelated stationary phase especially Fe³⁺ and Ga³⁺. The IMAC enriches the phosphorylated serine, threonine and tyrosine residues, because it is based on the presence of negatively charged phosphate group [187-189].

In general phosphopeptides are difficult to be analysed by mass spectrometry: (i), because they bear negative charge, whereas electrospray is normally operated in the positive mode, (ii), they are usually hydrophilic and therefore they do not bind well to the columns used for peptide separation prior MS analysis and hence, they can not be seen as an intense peak, because of ionic suppression by the non-phosphorylated peptide, which are also present, (iii), the peptides sizes are important as well, because they can not be observed in the spectrum, if the

enzyme used produces too large or too small peptide fragments (iv), mass spectrometric detection of phosphopeptides is not distributed to all possible amino acids residues target for phosphorylation modification. Thus, phosphotyrosine residues are more stable, while phosphoserine and phosphothreonine are lable (e. g.

β-elimination) [185]. In the present work high performance LTQ Orbitrap was used, which proved to be an excellent tool for determination of phosphoserine structure modification.

Figure 2.46. Identification of phosphorylation sites using LC-tandem mass spectrometry. Protein spot was cut out from the gel digested using trypsin and the resulting peptides were analysed by MS. Using electrospray ionization the peptide ion at m/z 942.959 (2+) was selected for CID fragmentation, which preferentially occurs at amide bond to generate C-terminal fragments (y ions) and N-terminal fragments (b ions) providing structural information regarding the amino acid sequence and phosphate moiety (80 Da) location (A), compared with unmodified peptide (B).

The phosphorylated structure present at the Ser-40 was determined from the LC-tandem MS analysis of peptides derived from in-gel digest with trypsin of 2-D gel protein spot. The phosphoserine was observed in the tryptic peptide ³³AAVPSGA STGIYEALELR⁵⁰ located at the N-terminal of beta-enolase full-length sequence. The identity of the peptide backbone and the exact localization of the phosphorylation site were determined from the MS/MS analyses of the doubly charged peptide ions using data-dependent acquisition, and from the mass differences between the observed phosphorylated peptide at m/z 942.959 (2+) and and unphosphorylated peptide at m/z 902.977 (2+) (Figure 2.46). Under CID fragmentation phosphopeptides do not only produce sequence specific fragments, but also fragments that are specific for phosphate group as they commonly undergo a gase-phase β-elimination reaction, resulting in a neutral loss of phosphoric acid (H3PO4) -98 Da or they are dephosphorylated (HPO3) – 80 Da; such situation is shown by y11 fragment ion at m/z 1215.2. Generally, phosphoserine and phosphothreonine may undergo an elimination reaction under CID, whereby phosphoric acid is lost and α, β-unsaturated bond is formed. The end products are dehydroalanine and dehydroaminobutyric acid, respectively. The loss of phosphate as HPO3 or H3PO4 is a dominant fragmentation event, over the backbone cleavages necessary for sequence determination. The phosphate loss may occur, mainly, when the parent ion is in the single charged state.

The identification of the phosphorylation site in this work was performed without any enrichment step. The difficulties encounted in such experiment come from the high complexity of peptide mixture, even if they belong to a single protein as two or more peptide (phosphorylated and unphosphorylated) may co-elute and, there is no time to obtain MS/MS spectra of each species. This situation was overcome by the use of HPLC peptide fractionation with long gradiednt in combination with high performance LTQ Orbitrap MS, operating repeated and fast scanning over the chromatographic peak, which improved the CID analysis and proved to be highly efficient for the identification of phosphorylation sites. The extent and variety of other modifications, a protein/peptide bears, besides phosphorylation may contitute another obstacle in the attempt of identification of phosphorylation sites. Figure 2.47 shows a further example of phosphorylation site located at Se-176 of full-length beta-enolase. The series of b and y ions gave the possibility to distinguish, which of the two serines are modified and the oxidation of Met-165 as well.

Figure 2.47. Double charged ion showing phosphorylation of beta-enolase protein at one of the two consecutive serines, Ser-176 and Ser-177. (A) Collision-indused Dissociation (CID) of the double charged parent ion at m/z 996.970 was chosen automatically in data-dependent mode. The CID fragments show amino acids sequence (163-179) with one phosphate. The presence of y3 excludes the possibility of phosphorylation at Ser-177. An increase in peptide mass with 16 Da besides the 80 Da representing phospho group compared to the unmodified peptide (B) is explained by methionine sulfoxide formation at Met-165 shown by the almost complete series of b ions.

2.6 Proteome analysis of nitration and related oxidatively modifications in