O-glycosylation of the non-canonical T-cadherin from rabbit skeletal muscle by single mannose residues

O-mannosylation is a vital protein modification. In humans, defective O-mannosylation of α-dystroglycan results in severe congenital muscular dystrophies. However, other proteins bearing this modification in vivo are still largely unknown. Here, we describe a highly reliable method combining glycosidase treatment with LC–MS analyses to identify mammalian O-mannosylated proteins from tissue sources. Our workflow identified T-cadherin (H-cadherin, CDH13) as a novel O-mannosylated protein. In contrast to known O-mannosylated proteins, single mannose residues (Man-α-Ser/Thr) are attached to this cell adhesion molecule. Conserved O-glycosylation sites in T-, E- and N-cadherins from different species, point to a general role of O-mannosyl glycans for cadherin function.     

LC–MS/MS characterization of combined glycogenin-1 and glycogenin-2 enzymatic activities reveals their self-glucosylation preferences

Glycogen synthesis is initiated by self-glucosylation of the glycosyltransferases glycogenin-1 and -2 that, in the presence of UDP-glucose, form both the first glucose-O-tyrosine linkage, and then stepwise add a series of α1,4-linked glucoses to a growing chain of variable length. Glycogen-1 and -2 coexist in liver glycogen preparations where the proteins are known to form homodimers, and they also have been shown to interact with each other. In order to study how glycogenin-1 and -2 interactions may influence each other's glucosylations we setup a cell-free expression system for in vitro production and glucosylation of glycogenin-1 and -2 in various combinations, and used a mass spectrometry based workflow for the characterization and quantitation of tryptic glycopeptides originating from glycogenin-1 and -2. The analysis revealed that the self-glucosylation endpoint was the incorporation of 4–8 glucose units on Tyr 195 of glycogenin-1, but only 0–4 glucose units on Tyr-228 of glycogenin-2. The glucosylation of glycogenin-2 was enhanced to 2–4 glucose units by the co-presence of enzymatically active glycogenin-1. Glycogenin-2 was, however, unable to glucosylate inactive glycogenin-1, at least not an enzymatically inactivated Thr83Met glycogenin-1 mutant, recently identified in a patient with severe glycogen depletion.     

Characterization of a TiO₂ enrichment method for label-free quantitative phosphoproteomics

Phosphorylation is a protein post-translational modification with key roles in the regulation of cell biochemistry and signaling. In-depth analysis of phosphorylation using mass spectrometry is permitting the investigation of processes controlled by phosphorylation at the system level. A critical step of these phosphoproteomics methods involves the isolation of phosphorylated peptides from the more abundant unmodified peptides produced by the digestion of cell lysates. Although different techniques to enrich for phosphopeptides have been reported, there are limited data on their suitability for direct quantitative analysis by MS. Here we report a TiO₂ based enrichment method compatible with large-scale and label-free quantitative analysis by LC–MS/MS. Starting with just 500 μg of protein, the technique reproducibly isolated hundreds of peptides, >85% of which were phosphorylated. These results were obtained by using relatively short LC–MS/MS gradient runs (45 min) and without any previous separation step. In order to characterize the performance of the method for quantitative analyses, we employed label-free LC–MS/MS using extracted ion chromatograms as the quantitative readout. After normalization, phosphopeptides were quantified with good precision (coefficient of variation was 20% on average, n = 900 phosphopeptides), linearity (correlation coefficients >0.98) and accuracy (deviations <20%). Thus, phosphopeptide ion signals correlated with the concentration of the respective phosphopeptide in samples, making the approach suitable for in-depth relative quantification of phosphorylation by label-free LC–MS/MS.     

Multiplexed quantification of 63 proteins in human urine by multiple reaction monitoring-based mass spectrometry for discovery of potential bladder cancer biomarkers

Three common urological diseases are bladder cancer, urinary tract infection, and hematuria. Seventeen bladder cancer biomarkers were previously discovered using iTRAQ - these findings were verified by MRM-MS in this current study. Urine samples from 156 patients with hernia (n=57, control), bladder cancer (n=76), or urinary tract infection/hematuria (n=23) were collected and subjected to multiplexed LC-MRM/MS to determine the concentrations of 63 proteins that are normally considered to be plasma proteins, but which include proteins found in our earlier iTRAQ study. Sixty-five stable isotope-labeled standard proteotypic peptides were used as internal standards for 63 targeted proteins. Twelve proteins showed higher concentrations in the bladder cancer group than in the hernia and the urinary tract infection/hematuria groups, and thus represent potential urinary biomarkers for detection of bladder cancer. Prothrombin had the highest AUC (0.796), with 71.1% sensitivity and 75.0% specificity for differentiating bladder cancer (n=76) from non-cancerous (n=80) patients. The multiplexed MRM-MS data was used to generate a six-peptide marker panel. This six-peptide panel (afamin, adiponectin, complement C4 gamma chain, apolipoprotein A-II precursor, ceruloplasmin, and prothrombin) can discriminate bladder cancer subjects from non-cancerous subjects with an AUC of 0.814, with a 76.3% positive predictive value, and a 77.5% negative predictive value. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.     

RP-HPLC-ESI-MS evidenced that salivary cystatin B is detectable in adult human whole saliva mostly as S-modified derivatives: S-Glutathionyl, S-cysteinyl and S-S 2-mer

An HPLC-ESI-MS analysis of adult human whole saliva evidenced three protein masses (M average 11,487 ± 2, 11,301 ± 2 and 22,362 ± 3 Da) eluting in the 32.5-35.0 min range. Treatment in reducing conditions allowed establishing that they are S-derivatives of N-terminal acetylated cystatin B, namely its S-glutathionyl, S-cysteinyl and S-S dimer. The identification was confirmed by high resolution HPLC-ESI-MS-MS experiments on the intact naturally occurring proteins and their tryptic digests. S-unmodified cystatin B is rarely detectable in whole saliva of healthy adults (5 subjects out of 65) and its percentage does not overcome approximately 20% of total cystatin B (11 ± 9%). In the majority of subjects (60 out of 65) the mean percentages of the S-modified derivatives were S-glutathionyl 53 ± 13%, S-cysteinyl 15 ± 5%, S-S 2-mer 32 ± 13%. Variations of the percentages of these S-modified derivatives of cystatin B could be indicative of oral oxidative stress. As we are aware, this is the first time that S-glutathionylation and S-cysteinylation were described as extensive PTM of a salivary protein and the first time that these PTMs were detected in naturally occurring cystatin B.     

A new β-chain haemoglobin variant with increased oxygen affinity: Hb Roma [β115(g17)Ala → Val]

Background

Haemoglobin Roma [β115(G17)Ala → Val] is a new adult haemoglobin variant found in a patient presenting a mild hypochromia and microcytosis. We studied this previously uncharacterised variant in order to evaluate the effect on the structural and funcional properties of the Ala → Val substitution at the α1β1 interface.

Methods and results

The variant chain was identified by direct DNA sequencing of the β-globin gene, which revealed a GCC → GTC mutation in codon 115. This mutation was confirmed by mass spectrometric analysis of the tetramers and peptides. The oxygen-binding properties of the haemoglobin A/haemoglobin Roma mixture, in which the variant makes up 25% of the haemoglobins, showed a significant increase in oxygen affinity with respect to normal haemoglobin A, both in the absence and presence of 2,3-bisphosphoglycerate. The role of the βG17 position, situated at the α₁β₁ interface, has been examined using computational models of haemoglobin Roma and other known βG17 variants, in comparison with normal haemoglobin A.

Conclusions

This study suggests that the β115(G17)Ala → Val substitution at the α1β1 interface is responsible for increased oxygen affinity and mild destabilisation of the haemoglobin Roma.

General significance

An amino acid substitution at the G17 position of the α1β1 interface may result in stabilisation of the high affinity R-state of the haemoglobin molecule.