Label-Free Quantitative Phosphoproteomics of the Fission Yeast Schizosaccharomyces pombe Using Strong Anion Exchange- and Porous Graphitic Carbon-Based Fractionation Strategies

Abstract

The phosphorylation of proteins modulates various functions of proteins and plays an important role in the regulation of cell signaling. In recent years, label-free quantitative (LFQ) phosphoproteomics has become a powerful tool to analyze the phosphorylation of proteins within complex samples. Despite the great progress, the studies of protein phosphorylation are still limited in throughput, robustness, and reproducibility, hampering analyses that involve multiple perturbations, such as those needed to follow the dynamics of phosphoproteomes. To address these challenges, we introduce here the LFQ phosphoproteomics workflow that is based on Fe-IMAC phosphopeptide enrichment followed by strong anion exchange (SAX) and porous graphitic carbon (PGC) fractionation strategies. We applied this workflow to analyze the whole-cell phosphoproteome of the fission yeast Schizosaccharomyces pombe. Using this strategy, we identified 8353 phosphosites from which 1274 were newly identified. This provides a significant addition to the S. pombe phosphoproteome. The results of our study highlight that combining of PGC and SAX fractionation strategies substantially increases the robustness and specificity of LFQ phosphoproteomics. Overall, the presented LFQ phosphoproteomics workflow opens the door for studies that would get better insight into the complexity of the protein kinase functions of the fission yeast S. pombe.

Mapping and Analysis of Swi5 and Sfr1 Phosphorylation Sites

“…In our previous work, we used the tandem affinity purification (TAP) protocol to purify Swi5-TAP and Sfr1-TAP from cycling S. pombe cells and found that Swi5 was phosphorylated on serine 72 and Sfr1 contained three phosphorylated serine residues (S26, S109 and S165) [ 24 ]. Additional phosphorylation sites on Sfr1 (S24, S26, T73, S109, S116 and S165) were published during the course of our work [ 25 , 26 , 27 , 28 , 29 ].…”

Identification of Nrl1 Domains Responsible for Interactions with RNA-Processing Factors and Regulation of Nrl1 Function by Phosphorylation

“…Previous studies have shown that many splicing factors have to be phosphorylated in order to function properly [8,. Although previous studies showed that Nrl1 is phosphorylated on multiple sites (T26, S122, S131 and S970) [49][50][51][52][53], little was known about the importance of these post-translational modifications. Here, by analyzing the isolated Nrl1 complexes we found that Nrl1 is also phosphorylated on residues S86 and S112 (Figure 5).…”

“…Previous studies have shown that Nrl1 is phosphorylated on several amino acid residues [42,45,[49][50][51][52][53]. This suggested that, similarly to other splicing factors, the function of Nrl1 might be regulated by phosphorylation.…”

Advances in proteomics sample preparation and enrichment for phosphorylation and glycosylation analysis

“…for PTMs protein sample preparation and enrichment. For example, the enrichment strategies operated in phosphoproteomic studies are molecularly imprinted technology (MIP) [25,26], ion exchange chromatography (IEC) [27][28][29], affinity chromatography [30][31][32], and chemical derivatization [33,34]. Different from phosphorylation modifications with fixed structures, the diversity of polysaccharides and the interference of glycans on the peptide backbone make the identification of glycopeptides and the study of protein glycosylation encounter more obstacles.…”