Liquid Chromatography Methods for Analysis of mRNA Poly(A) Tail Length and Heterogeneity.

Messenger RNA (mRNA) is a new class of therapeutic compounds. The current advances in mRNA technology require the development of efficient analytical methods. In this work, we describe the development of several methods for measurement of mRNA poly(A) tail length and heterogeneity. Poly(A) tail was first cleaved from mRNA with the RNase T1 enzyme. The average length of a liberated poly(A) tail was analyzed with the size exclusion chromatography method. Size heterogeneity of the poly(A) tail was estimated with high-resolution ion-pair reversed phase liquid chromatography (IP RP LC). The IP RP LC method provides resolution of poly(A) tail oligonucleotide variants up to 150 nucleotide long. Both methods use a robust ultraviolet detection suitable for mRNA analysis in quality control laboratories. The results were confirmed by the LC-mass spectrometry (LC MS) analysis of the same mRNA sample. The poly(A) tail length and heterogeneity results were in good agreement.

Characterization of Glycoproteoforms of Integrins α2 and ß1 in Megakaryocytes in the Occurrence of JAK2V617F Mutation-Induced Primary Myelofibrosis.

Primary myelofibrosis (PMF) is a neoplasm prone to leukemic transformation, for which limited treatment is available. Among individuals diagnosed with PMF, the most prevalent mutation is the JAK2V617F somatic point mutation that activates the Janus kinase 2 (JAK2) enzyme. Our earlier reports on hyperactivity of ß1 integrin and enhanced adhesion activity of the α2ß1 complex in JAK2V617F megakaryocytes (MKs) led us to examine the new hypothesis that this mutation leads to posttranslational modification via changes in glycosylation. Samples were derived from immunoprecipitation of MKs obtained from Vav1-hJAK2V617F and WT mice. Immunoprecipitated fractions were separated by SDS-PAGE and analyzed using LC-MS/MS techniques in a bottom-up glycoproteomics workflow. In the immunoprecipitate, glycopeptiforms corresponding to 11 out of the 12 potential N-glycosylation sites of integrin ß1 and to all nine potential glycosylation sites of integrin α2 were observed. Glycopeptiforms were compared across WT and JAK2V617F phenotypes for both integrins. The overall trend observed is that JAK2V617F mutation in PMF MKs leads to changes in ß1 glycosylation; in most cases, it results in an increase in the integrated area of glycopeptiforms. We also observed that in mutated MKs, changes in integrin α2 glycosylation were more substantial than those observed for integrin ß1 glycosylation, a finding that suggests that altered integrin α2 glycosylation may also affect activation. Additionally, the identification of proteins associated to the cytoskeleton that were co-immunoprecipitated with integrins α2 and ß1 demonstrated the potential of the methodology employed in this study to provide some insight, at the peptide level, into the consequences of integrin activation in MKs. The extensive and detailed glycosylation patterns we uncovered provide a basis for future functional studies of each site in control cells as compared to JAK2V617F-mutated cells. Data are available via ProteomeXchange with identifier PXD030550.

Respiratory Selenite Reductase from Bacillus selenitireducens Strain MLS10.

The putative respiratory selenite [Se(IV)] reductase (Srr) from Bacillus selenitireducens MLS10 has been identified through a polyphasic approach involving genomics, proteomics, and enzymology. Nondenaturing gel assays were used to identify Srr in cell fractions, and the active band was shown to contain a single protein of 80 kDa. The protein was identified through liquid chromatography-tandem mass spectrometry (LC-MS/MS) as a homolog of the catalytic subunit of polysulfide reductase (PsrA). It was found to be encoded as part of an operon that contains six genes that we designated srrE, srrA, srrB, srrC, srrD, and srrF SrrA is the catalytic subunit (80 kDa), with a twin-arginine translocation (TAT) leader sequence indicative of a periplasmic protein and one putative 4Fe-4S binding site. SrrB is a small subunit (17 kDa) with four putative 4Fe-4S binding sites, SrrC (43 kDa) is an anchoring subunit, and SrrD (24 kDa) is a chaperon protein. Both SrrE (38 kDa) and SrrF (45 kDa) were annotated as rhodanese domain-containing proteins. Phylogenetic analysis revealed that SrrA belonged to the PsrA/PhsA clade but that it did not define a distinct subgroup, based on the putative homologs that were subsequently identified from other known selenite-respiring bacteria (e.g., Desulfurispirillum indicum and Pyrobaculum aerophilum). The enzyme appeared to be specific for Se(IV), showing no activity with selenate, arsenate, or thiosulfate, with a Km of 145 ± 53 µM, a Vmax of 23 ± 2.5 µM min-1, and a kcat of 23 ± 2.68 s-1 These results further our understanding of the mechanisms of selenium biotransformation and its biogeochemical cycle.IMPORTANCE Selenium is an essential element for life, with Se(IV) reduction a key step in its biogeochemical cycle. This report identifies for the first time a dissimilatory Se(IV) reductase, Srr, from a known selenite-respiring bacterium, the haloalkalophilic Bacillus selenitireducens strain MLS10. The work extends the versatility of the complex iron-sulfur molybdoenzyme (CISM) superfamily in electron transfer involving chalcogen substrates with different redox potentials. Further, it underscores the importance of biochemical and enzymological approaches in establishing the functionality of these enzymes.

Multiple solution structures of the disordered peptide indolicidin from IMS-MS analysis.

The solution-favored conformations of the 13-residue disordered peptide, indolicidin (Ile1-Leu2-Pro3-Trp4-Lys5-Trp6-Pro7-Trp8-Trp9-Pro10-Trp11-Arg12-Arg13), are evaluated using electrospray ionization (ESI) coupled to ion mobility spectrometry-mass spectrometry (IMS-MS). The ESI-IMS-MS distributions for the dominant [M+4H]4+ ions indicate that three populations of structures coexist in a range of aqueous to non-aqueous solutions (water:dioxane, water:trifluoroethanol, and water:hexafluoroisopropanol). Conformer types and their relative abundances change in response to different solution environments suggesting that the gas phase conformers reflect on the solution populations present in different solvent environments. Collisional activation of isolated gas phase conformations with IMS-IMS-MS experiments provides additional insight about the relative stabilities of different structural types in the absence of solvent. Simulated annealing studies suggest that proline configuration may be important for the presence of multiple conformations.

Delineating diseases by IMS-MS profiling of serum N-linked glycans.

Altered branching and aberrant expression of N-linked glycans is known to be associated with disease states such as cancer. However, the complexity of determining such variations hinders the development of specific glycomic approaches for assessing disease states. Here, we examine a combination of ion mobility spectrometry (IMS) and mass spectrometry (MS) measurements, with principal component analysis (PCA) for characterizing serum N-linked glycans from 81 individuals: 28 with cirrhosis of the liver, 25 with liver cancer, and 28 apparently healthy. Supervised PCA of combined ion-mobility profiles for several, to as many as 10 different mass-to-charge ratios for glycan ions, improves the delineation of diseased states. This extends an earlier study [J. Proteome Res.2008, 7, 1109-1117] of isomers associated with a single glycan (S(1)H(5)N(4)) in which PCA analysis of the IMS profiles appeared to differentiate the liver cancer group from the other samples. Although performed on a limited number of test subjects, the combination of IMS-MS for different combinations of ions and multivariate PCA analysis shows promise for characterizing disease states.