Why phosphoproteomics is still a challenge

Abstract

Despite continuous improvements phosphoproteomics still faces challenges that are often neglected, e.g. partially poor recovery of phosphopeptide enrichment, assessment of phosphorylation stoichiometry, label-free quantification, poor behavior during chromatography, and general limitations of peptide-centric proteomics. Here we critically discuss current limitations that need consideration in both qualitative and quantitative studies.

Simple, scalable, and ultrasensitive tip-based identification of protease substrates

“…Nevertheless, combined with optimal sampling strategies and standardization, the here presented sensitivity may even allow Nterminomics of needle biopsies. We also analyzed our data for potential physicochemical biases that may arise from the enrichment as this is not uncommon for PTM-enrichment procedures (36). We therefore compared the 3,086 unique identified N-terminal peptides identified in this study to N-terminal peptides in the Peptide Atlas (October 2017, 1,222,862 peptide entries in total), as described in the Supplemental…”

Joining forces: studying multiple post-translational modifications to understand dynamic disease mechanisms

“…This comes along with a variety of shortcomings as discussed elsewhere [11]. Nevertheless, peptides share much less heterogeneous characteristics than proteins and they can be more easily analyzed/ identified by liquid chromatography-mass spectrometry.…”

Proteomic characterization of the acid-insoluble fraction of whole saliva from preterm human newborns

“…Very often this pattern is associated to protein species differing in their degree of phosphorylation, however, under our experimental conditions we were unable to identify phosphorylated peptides after in-gel tryptic digestion and MS/MS analysis. It should be pointed out that presence of phosphate groups is not easily evidenced by MS/MS due to a number of factors [12] including the labile phosphate group upon collision induced dissociation leading to the hydrolysis of the phosphate group linked to Ser/Thr residues [13], the reduced ionisation efficiency of phosphopeptides due to the phosphate group negative charge, the reduction of tryptic cleavage within phosphorylated motifs [14].…”

“…Among them, cytoskeletal keratin 13, belonging to type I (67, 69, 94), and keratin 4 type II (66, 74, 75, 78), annexin A1 (31, 32) and annexin A2 (36, 83), actin cytoplasmic 1 (88), serpin B3 (33) and serpin B5 (89), glyceraldehyde-3-phosphate dehydrogenase (37), alpha enolase (35), serum albumin (92) and cornulin (72, 73) were identified. Among the proteins localized in the lower part of the gel, we identified S100A8 (8,9) and S100A9 (1, 2 and 11), cystatin B (6), thioredoxin (12), epidermal fatty acid-binding protein (E-FABP) in 3, galectin-7 (81), and calmodulin-like protein 3 (14).…”