2.2. Literature review of Saccharomyces cerevisiae proteomic analysis
2.2.5. Proteomic analysis of S. cerevisiae protein modifications
Phosphorylation is one of the most common techniques for the post-translational modification of S. cerevisiae proteins. The identification of phosphorylation sites of proteins based using a MS workflow has been carried out [87]. However, to understand the biological function of phosphorylation, the quantitative analysis of phosphorylation in a site specific manner is necessary, and early applications of this approach were performed using SILAC (using 15N-labeled enriched media) techniques [88, 89]. In an attempt to increase the ionization efficiency and CID of the labeled peptides for identification and quantitation by MS, the N-isotag, in which a stable isotope-coded amino acids react with phosphopeptides and PTM (post-translational modification) peptides, was developed by Smolka [82].
One of the most important factors in the study of a proteome is the sensitivity in detecting peptides and then proteins, especially modified proteins in complex samples. To meet this requirement, some techniques can be used such as fractionating organelles, isolating specific modified proteins (tyrosine phosphorylated proteins). Centrifugation with different solubility, or antibody directed methods can be used to improve the fractionation of organelles [90]. The improvement of fractionation resulted in increased numbers (up to 750) of proteins identified in S. cerevisiae mitochondria [54]. The enrichment of phosphopeptides using immobilized metal affinity chromatography (IMAC), and sufficient starting material helped in the detection of more than 700 phosphopeptides in S. cerevisiae [91].
Low-energy collision-induced dissociation (CID) was used to fragment the peptide backbone and to produce ions of type’s b and y phosphorylation that is required for successful sequence analysis and identification of phosphorylation sites [92]. However, of 1,000 phosphopeptides detected, only 383 sites of phosphorylation were identified because the CID mode often promoted elimination of phosphoric acid from serine and threonine
residues without breaking the amide bonds along the peptide backbone. The resulting MS/MS spectra were devoid of sequence information [93]. To overcome this problem, in 2004, Syka et al. developed a new method called electron transfer dissociation (ETD) for the fragmentation of peptides (proteins) [94]. The release of this innovative new fragmentation technology, by a range of vendors including Bruker Daltoniks, Agilent and Thermo Scientific has offered a new generation MS for studying proteomes, especially for PTMs, since amino acid side chains and important modifications, such as phosphorylation, are left intact when using this technique [95, 96]. Recently, the analysis of the yeast phosphoproteome using endo-Lys C as the proteolytic enzyme, IMAC for phosphopeptide enrichment, and ETD was applied by Chi et al., and as a result, 1,252 phosphorylation sites on 629 proteins were identified in sample containing 30 μg of whole cell lysate [93].
However, peptides with charges of +3 or higher are required for effective fragmentation, whilst small peptides with predominantly +2 charges gave poorer fragmentation efficiency with ETD or ECD [96, 97].
Other types of protein modifications have also been analysed in S. cerevisiae, including ubiquitination [98], and protein degradation [99]. Peng et al. applied a technique for the large scale analysis and characterization of protein ubiquitination. In this study, ubiquitin conjugated from a strain expressing 6xHis-tagged ubiquitin were isolated, trypsin digested, and then analysed by tandem MS/MS. A total of 1,075 proteins and 110 precise ubiquitination sites were detected [98]. Additionally, a study of protein degradation in S.
cerevisiae was carried out by Bell et al., where the half-lives of more than 3,750 S.
cerevisiae proteins were measured using an epitope-tagged strain collection [99].
An observation of 79.97 m/z (HPO4) mass difference in the MS spectrum is an initial indicator for a phosphopeptide in a protein digest. In the negative ion mode, marker ions (from phosphotpeptide) at a m/z 96.97 (H2PO4-) and 78.96 (PO3-) are observed, which can be used for their recognition by precursor ion scanning (PIS) [100], or skimmer CID
LC/ESI-MS [101]. The current MS instruments are designed to perform neither for simultaneous detection of negative and positive ions nor for fast polarity switching, however, MS instruments are strongly preferred [102]. In standard positive ion polarity, observation of a neutral loss of 97.97 m/z (H3PO4) is the method of choice for recognition of pS- and pT-phosphopeptides [103], whilst for pY peptides, a characteristic loss of 79.97 m/z (HPO3) is observed [104] and a specific pY immonium ion at 216.04 is formed.
Subsequently, high-resolution precursor ion scanning (performed in Q-TOF-MS) was used for specific detection of pY peptides from phosphoproteins enriched using an immobilized anti-pY antibody [105]. Recently, two different MS analyses were used to identify the phophorylated residues; where while a data-dependent neutral loss analyses was used to fragment peptides to enhance the neutral loss of phosphoric acid, a targeted product ion scanning (TPIS) mass analysis was performed where MS2 are triggered for specific m/Z values [106].
IMAC was also used in another study carried out by Ficarro et al., in which more than 1,000 phosphopeptides were detected from the whole cell lysate of S. cerevisiae. Among these phosphopeptides, 216 were detected defining 383 sites of phosphorylation, of which 60, 145 and 11 were singly, doubly, and triply phosphorylated, respectively [107]. In an attempt to rapidly increase the identification of large numbers of samples for PTM investigations, protein chips were developed and used to examine protein kinases in S.
cerevisiae [108]. This technique proved useful for high-throughput screening of protein biochemical activity since nearly all (119) of the yeast protein kinases were used to analyse specificity using 17 different substrates, leading to 32 kinases preferentially phosphorylating one or two substrates, and 27 kinases readily phosphorylating in more than three substrates were determined [108].
Although new proteomics technologies offer a great benefit with high throughput identification of large numbers of proteins, the current methods for the identification of
PTMs (for example phosphorylation) are still insensitive. Therefore, the improvement of sensitivities using new MS technologies for identification of S. cerevisiae proteins offers a new view on the identification of large number of proteins in response to a specific state.