Determining disease-associated changes in protein glycosylation provides a better understanding of pathogenesis. This work focuses on human immunoglobulin A1 (IgA1), where aberrant O-glycosylation plays a key role in the pathogenesis of IgA nephropathy (IgAN). Normal IgA1 hinge region carries 3 to 6 O-glycans consisting of N-acetylgalactosamine (GalNAc) and galactose (Gal); both sugars may be sialylated. In IgAN patients, some O-glycans on a fraction of IgA1 molecules are Gal-deficient. Here we describe a sample preparation protocol with optimized cysteine alkylation of a Gal-deficient polymeric IgA1 myeloma protein prior to in-gel digestion and analysis of the digest by MALDI-TOF/TOF mass spectrometry (MS). Following a novel strategy, IgA1 hinge-region O-glycopeptides were fractionated by reversed-phase liquid chromatography using a microgradient device and identified by MALDI-TOF/TOF tandem MS (MS/MS). The acquired MS/MS spectra were interpreted manually and by means of our own software. This allowed assigning up to six O-glycosylation sites and demonstration, for the first time, of the distribution of isomeric O-glycoforms having the same molecular mass, but a different glycosylation pattern. The most abundant Gal-deficient O-glycoforms were GalNAc4Gal3 and GalNAc5Gal4 with one Gal-deficient site and GalNAc5Gal3 and GalNAc4Gal2 with two Gal-deficient sites. The most frequent Gal-deficient sites were at Ser230 and/or Thr236.
An acidic prolyl endoprotease from Aspergillus niger was isolated from the commercial product Brewers Clarex to evaluate its possible application in proteomics. The chromatographic purification yielded a single protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis providing an apparent molecular mass of 63 kDa and a broad peak (m/z 58,061) in linear matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) indicating the glycoprotein nature of the enzyme. Indeed, a colorimetric assessment with phenol and sulfuric acid showed the presence of neutral sugars (9% of weight). The subsequent treatment with N-glycosidase F released a variety of high-mannose type N-glycans, which were successfully detected using MALDI-TOF MS. MALDI-TOF/TOF tandem MS analysis of glycopeptides from a tryptic digest of prolyl endoprotease unraveled the identity of the N-glycosylation site in the primary structure. The data obtained also show that the enzyme is present in its processed form, i.e. without putative signal and propeptide parts. Spectrophotometric measurements demonstrated optimal activity at pH 4.0-4.5 and also high thermostability for the cleavage at the C-terminal part of proline residues. In-solution digestion of standard proteins (12-200 kDa) allowed to evaluate the cleavage specificity. The enzyme acts upon proline and alanine residues, but there is an additional minor cleavage at some other residues like Gly, Leu, Arg, Ser and Tyr. The digestion of a honeybee peptide comprising six proline residues (apidaecin 1A) led to the detection of specific peptides terminated by proline as it was confirmed by MALDI postsource decay analysis.
Cyanobacteria represent a bacterial phyllum characteristic by the ability to photosynthesize. They are potentially applicable for the production of useful compounds but may also cause poisoning or at least health problems as they can produce cyanotoxins. The introduction of a fast methodology is important not only for fundamental taxonomic purposes, but also for reliable identifications in biological studies. In this work, we have used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of intact cells to study Chroococcidiopsis strains. A library of the obtained reference mass spectra containing characteristic peptide/protein profiles was examined by software tools to characterize similarities and differences applicable for diagnostics and taxonomy. Both a similarity tree and heat map constructed from the mass spectrometric data proved consistent with 16S rRNA sequencing results. We show as novelty that a binary matrix combining ferulic and sinapinic acids performs well in acquiring reproducible mass spectra of cyanobacteria. Using the matrix solvent, a protein extraction from cells was done. After polyacrylamide gel electrophoresis, the separated protein fractions were in-gel digested and the resulting peptides analyzed by liquid chromatography coupled with tandem mass spectrometry. For the first time, photosystem protein components, phycobilisome proteins, electron transport proteins, nitrogen-metabolism and nucleic acids binding-proteins, cytochromes plus other enzymes and various uncharacterized proteins could be assigned to characteristic peaks in the mass spectrometric profiles and some of them suggested as markers in addition to 30S and 50S ribosomal proteins known from previous studies employing intact cell mass spectrometry of microorganisms.
Trypsin is a protease, which is commonly used for the digestion of protein samples in proteomic experiments. The process of trypsin autolysis is known to produce autolytic peptides as well as active enzyme forms with one or more intra-chain splits. In consequence, their variable presence can influence the digestion of a protein substrate in the reaction mixture. Besides two major and well-studied forms named β-trypsin and α-trypsin, there are also other active trypsin forms known such as γ-trypsin and pseudotrypsin (ψ-trypsin). In this work, the cleavage specificity of ψ-trypsin was evaluated using in-gel digestion of protein standards followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analyses of the resulting peptides. The numbers of produced and matching peptides were similar to those obtained using α-/β-trypsin. The same experience was obtained with a real complex protein sample from rat urine. In previous reports, ψ-trypsin was supposed to generate non-specific cleavages, which has now been reevaluated. Purified ψ-trypsin cleaved all analyzed proteins preferentially on the C-terminal side of Lys and Arg residues in accordance with the canonical tryptic cleavage. However, a minor nonspecific cleavage performance was also registered (particularly after Tyr and Phe), which was considerably higher than in the case of trypsin itself.
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