Phosphospecific proteolysis for mapping sites of protein phosphorylation

Abstract

Protein phosphorylation is a dominant mechanism of information transfer in cells, and a major goal of current proteomic efforts is to generate a system-level map describing all the sites of protein phosphorylation. Recent efforts have focused on developing technologies for enriching and quantifying phosphopeptides. Identification of the sites of phosphorylation typically relies on tandem mass spectrometry to sequence individual peptides. Here we describe an approach for phosphopeptide mapping that makes it possible to interrogate a protein sequence directly with a protease that recognizes sites of phosphorylation. The key to this approach is the selective chemical transformation of phosphoserine and phosphothreonine residues into lysine analogs (aminoethylcysteine and beta-methylaminoethylcysteine, respectively). Aminoethylcysteine-modified peptides are then cleaved with a lysine-specific protease to map sites of phosphorylation. A blocking step enables single-site cleavage, and adaptation of this reaction to the solid phase facilitates phosphopeptide enrichment and modification in one step.

Phosphorylation of the FUS low‐complexity domain disrupts phase separation, aggregation, and toxicity

“…None of the four conserved TQ sites were detected. We and others have suspected that the complete lack of positively charged residues in FUS LC obscures phosphorylation detection by standard proteomic workflows due to lack of positive residues both for efficient positive ion formation for mass spectrometry and for trypsin cleavage sites (Knight et al , 2003). Therefore, to determine the full possible extent of the FUS LC phosphorylation by DNA‐PK, we used in vitro DNA‐PK phosphorylation as described previously (Gardiner et al , 2008; Han et al , 2012; Deng et al , 2014) and detected phosphosites by two‐dimensional solution NMR, which offers direct residue resolution.…”

Analytical strategies for phosphoproteomics

“…By subsequently adding a chemical compound containing a free sulfhydryl group to dehydroalanine or b-methyldehydroalanine acid by Michael addition, a di-thiol is created in the peptide, which can serve as a cross-linker to a biotin-tag [98] or for direct affinity purification [99]. Knight and co-workers used the combination of b-elimination, Michael addition reaction and proteolysis to aid the assignment of phosphorylation sites using tandem MS analysis [100]. In this study, phosphoserine and phosphothreonine were converted to aminoethylcysteine and b-methylaminoethylcysteine, respectively, both targets for lysine specific proteases (e.g., trypsin, and lysyl endopeptidase (Lys-C)).…”

State-of-the-art in phosphoproteomics

“…Furthermore, the chosen label can also alter the protein/peptide behaviour during sample preparation. Addition of cysteamine as a nucleophile introduces a positive charge into the protein generating a new tryptic cleavage site [72,73]. Similarly a reaction with dimethyl-aminoethanethiol and subsequent oxidation yields the possibility for positive mode precursor ion scanning for 2-dimethylaminoethanesulfoxide at m/z = 122.06 Da obtained by low energy CID [74].…”