Strong anion exchange-mediated phosphoproteomics reveals extensive human non-canonical phosphorylation.

Phosphorylation is a key regulator of protein function under (patho)physiological conditions, and defining site-specific phosphorylation is essential to understand basic and disease biology. In vertebrates, the investigative focus has primarily been on serine, threonine and tyrosine phosphorylation, but mounting evidence suggests that phosphorylation of other "non-canonical" amino acids also regulates critical aspects of cell biology. However, standard methods of phosphoprotein characterisation are largely unsuitable for the analysis of non-canonical phosphorylation due to their relative instability under acidic conditions and/or elevated temperature. Consequently, the complete landscape of phosphorylation remains unexplored. Here, we report an unbiased phosphopeptide enrichment strategy based on strong anion exchange (SAX) chromatography (UPAX), which permits identification of histidine (His), arginine (Arg), lysine (Lys), aspartate (Asp), glutamate (Glu) and cysteine (Cys) phosphorylation sites on human proteins by mass spectrometry-based phosphoproteomics. Remarkably, under basal conditions, and having accounted for false site localisation probabilities, the number of unique non-canonical phosphosites is approximately one-third of the number of observed canonical phosphosites. Our resource reveals the previously unappreciated diversity of protein phosphorylation in human cells, and opens up avenues for high-throughput exploration of non-canonical phosphorylation in all organisms.

Gas-phase intermolecular phosphate transfer within a phosphohistidine phosphopeptide dimer.

The hydrogen bonds and electrostatic interactions that form between the protonated side chain of a basic residue and the negatively charged phosphate of a phosphopeptide can play crucial roles in governing their dissociation pathways under low-energy collision-induced dissociation (CID). Understanding how phosphoramidate (i.e. phosphohistidine, phospholysine and phosphoarginine), rather than phosphomonoester-containing peptides behave during CID is paramount in investigation of these problematic species by tandem mass spectrometry. To this end, a synthetic peptide containing either phosphohistidine (pHis) or phospholysine (pLys) was analyzed by ESI-MS using a Paul-type ion trap (AmaZon, Bruker) and by traveling wave ion mobility-mass spectrometry (Synapt G2-Si, Waters). Analysis of the products of low-energy CID demonstrated formation of a doubly 'phosphorylated' product ion arising from intermolecular gas-phase phosphate transfer within a phosphopeptide dimer. The results are explained by the formation of a homodimeric phosphohistidine (pHis) peptide non-covalent complex (NCX), likely stabilized by the electrostatic interaction between the pHis phosphate group and the protonated C-terminal lysine residue of the peptide. To the best of our knowledge this is the first report of intermolecular gas-phase phosphate transfer from one phosphopeptide to another, leading to a doubly phosphorylated peptide product ion.

Identifying Developmental Language Disorder in Deaf Children with Cochlear Implants: A Case Study of Three Children.

(1) Background: While spoken language learning delays are assumed for deaf and hard of hearing (DHH) children after cochlear implant (CI), many catch up with their hearing peers. Some DHH children with CIs, however, show persistent delays in language, despite protective factors being in place. This suggests a developmental language disorder (DLD). However, at present there is little consensus on how to diagnose DLD in DHH children. (2) Methods: Given the lack of consensus in this area, a set of case studies provides an appropriate first step. The goal of this paper is to show the plausibility of a DLD diagnosis, following careful analysis of protective and risk factors. A retrospective case study review was conducted for three children. Their long-term language outcomes up to four years after CI were considered in the context of access to sound, speech sound discrimination, social skills and non-verbal cognition. (3) Results: It was possible to posit DLD in one child who had experienced good access to sound, alongside good speech discrimination abilities and social development, and normal non-verbal cognition, but who presented with severe language learning difficulties. (4) Conclusions: Finding markers for DLD in DHH children is important for diagnosis and intervention. The implications for clinical practice are discussed.

Pre-linguistic social communication skills and post implant language outcomes in deaf children with cochlear implants.

PURPOSE: This study investigates the relationship between pre-linguistic social communication skills and age of cochlear implant for future language outcomes in a large sample of deaf children. METHOD: A retrospective cohort study of records from 75 children. Pre-implant data included Age at Implant, pre-linguistic communication (social, symbolic and speech) skills, and non-verbal ability. Receptive and expressive language development data at 1 year, 2 years and 4 years post implant were analysed to investigate the relationships between pre-implant factors and language outcomes, in particular pre-linguistic social communication skills in early and late implanted children. RESULTS: Age at Implant was the strongest correlate of post implant expressive and receptive language outcomes. The sample was divided into early implanted (<18 month) and late implanted (>18 months) children. In the early implanted group, pre-linguistic social communication skills were the strongest pre-implant correlate of language outcomes four years post-implant. In the late implanted group, there were no significant pre-implant correlates of language outcomes. CONCLUSIONS: Long term language outcomes after cochlear implantation are the product of a set of communicative, cognitive and environmental factors. Early pre-implant social communication skills are an important consideration for clinicians who guide parents as to likely long-term outcomes post cochlear implantation. Social communication skills are particularly important for children who receive implants before the age of 18 months.

High-Throughput Characterization of Histidine Phosphorylation Sites Using UPAX and Tandem Mass Spectrometry.

Liquid chromatography (LC)-tandem mass spectrometry (MS/MS) is key for the characterization of phosphorylation sites in a high-throughput manner, and its application has proven essential to elucidate the phosphoproteome of many biological systems. Following proteolytic digestion of proteins extracted from tissues or cells, phosphopeptides are typically enriched by affinity chromatography using TiO2 or metal-ions (e.g., Fe3+) coupled to solid-phase materials, prior to LC-MS/MS analysis. Separation of relatively low abundance phosphopeptides from nonphosphorylated peptides in these types of extremely complex mixtures is essential to maximize coverage of the phosphoproteome. Maintaining acidic conditions during these IMAC or TiO2-based enrichment minimizes the concurrent unwanted binding of highly acidic peptides. However, while peptides containing phosphomonoesters, namely, phosphoserine (pSer), phosphothreonine (pThr), and phosphotyrosine (pTyr), are stable under these acidic binding conditions, phosphopeptides containing acid-labile phosphate group such as phosphohistidine (pHis), are not. Consequently, hydrolysis of these types of phosphopeptides occurs during standard phosphopeptide enrichment, and subsequent phosphosite identification by LC-MS/MS is severely compromised. Here we describe UPAX, unbiased phosphopeptide enrichment using strong anion exchange, for the separation of both acid-stable (pSer, pThr, pTyr) and acid-labile phosphopeptides (including those containing pHis) from nonphosphorylated peptides. We outline how implementation of UPAX prior to a minimally modified standard proteomics workflow can be used to identify sites of pHis as well as other acid-labile, as well as acid-stable phosphosites.

Myoblast Phosphoproteomics as a Tool to Investigate Global Signaling Events During Myogenesis.

Protein phosphorylation is a universal covalent chemical modification of amino acids involved in a large number of biological processes including cell signaling, metabolism, proliferation, differentiation, survival/death, ageing, and many more. Regulation of protein phosphorylation is essential in myogenesis and indeed, when the enzymatic activity of protein kinases is distrupted in myoblasts, myogenesis is affected. In this chapter we describe a method to profile the phosphoproteome of myoblasts using mass spectrometry. Phosphate groups are labile and easily lost during the processing of samples for mass spectrometry. Thus, effective methods to enrich for phosphopeptides from protein extracts have been developed. Here, we discuss and present in detail two such methods that we routinely employ. These methods are based on a sample enrichment step performed on titanium dioxide matrices followed by label-free tandem mass spectrometry and semi-quantitation.