Israeli acute paralysis virus infection leads to an enhanced RNA interference response and not its suppression in the bumblebee Bombus terrestris

RNA interference (RNAi) is the primary antiviral defense system in insects and its importance for pollinator health is indisputable. In this work, we examined the effect of Israeli acute paralysis virus (IAPV) infection on the RNAi process in the bumblebee, Bombus terrestris, and whether the presence of possible functional viral suppressors could alter the potency of the host's immune response. For this, a two-fold approach was used. Through a functional RNAi assay, we observed an enhancement of the RNAi system after IAPV infection instead of its suppression, despite only minimal upregulation of the genes involved in RNAi. Besides, the presence of the proposed suppressor 1A and the predicted OrfX protein in IAPV could not be confirmed using high definition mass spectrometry. In parallel, when bumblebees were infected with cricket paralysis virus (CrPV), known to encode a suppressor of RNAi, no increase in RNAi efficiency was seen. For both viruses, pre-infection with the one virus lead to a decreased replication of the other virus, indicating a major effect of competition. These results are compelling in the context of Dicistroviridae in multi-virus/multi-host networks as the effect of a viral infection on the RNAi machinery may influence subsequent virus infections

Pitfalls in histone propionylation during bottom-up mass spectrometry analysis

Despite their important role in regulating gene expression, posttranslational histone modifications remain technically challenging to analyze. For identification by bottom-up MS, propionylation is required prior to and following trypsin digestion. Hereby, more hydrophobic peptides are generated enabling RP HPLC separation. When histone dynamics are studied in a quantitative manner, specificity, and efficiency of this chemical derivatization are crucial. Therefore we examined eight different protocols, including two different propionylation reagents. This revealed amidation (up to 70%) and methylation (up to 9%) of carboxyl groups as a side reaction. Moreover, incomplete (up to 85%) as well as a specific propionylation (up to 63%) can occur, depending on the protocol. These results highlight the possible pitfalls and implications for data analysis when doing bottom-up MS on histones

Neutrophil elastase is the 'histone H2A-specific protease'

Fundamental changes in the epigenetic status of histones from hematopoietic stem cells might be one of the driving forces behind many malignant transformations and subsequent leukemia development. The amino-terminal tail of histones and the carboxy-tail of histone H2A protrude from the nucleosome and can be modified by many different posttranslational modifications (PTM) on at least 60 different residues, thereby mediating chromatin dynamics. During an iTRAQ proteome analysis on Chronic Lymphocytic Leukemia (CLL) B-cells we came across a specific kind of histone modification that has received only little attention in epigenetics until now: histone clipping. The clipping of the histone H2A C-tail at V114 was more abundant in the CLL B-cell clones compared to healthy B-cells. This specific proteolytic product was already described in the context of leukemia in the late 70’s and is still being referenced today. To specifically quantify this clipping product, we developed and optimized a sensitive and high throughput AQUA approach, based on two isotopically labeled synthetic peptides. We screened 36 patients to investigate any discriminative power of clipped H2A as a potential prognostic marker. In doing so, we found that clipping mainly occurs in the myeloid lineage and has no clear link to the CLL B-cell clone. Here we show that the responsible enzyme, until now known as the “H2A specific protease”, but previously not identified, actually is Neutrophil Elastase. With the growing interest in the epigenetic potential of histone clipping we emphasize its potential role in hematopoietic differentiation

Tryptic peptide analysis of protein binders in works of art by liquid chromatography–tandem mass spectrometry

Comprehension of the materials, such as binding media, used by artists is of uttermost importance in restoration and in art historical studies. The most frequently used binders are drying oils and proteins; in this study focus is placed on proteins. Most actual methods for protein binder identification are based on complete hydrolyzation of the protein matter into its amino acids and separation/detection with gas chromatography – mass spectrometry (GC-MS) or high performance liquid chromatography (HPLC) after derivatization. Because amino acids itself are not characteristic for a protein, identification is often based on the relative amount of 7 stable amino acids. In the current study a proteomics approach was used, in which the proteins were digested enzymatically into peptides using trypsin before being separated and detected by liquid chromatography – electronspray ionisation tandem mass spectrometry (LC-ESI-MS/MS). Mascot (Matrix Science) was used to analyze the resulting data and for protein identification. This way, amino acid sequences could be studied that retain much more information about the proteins, their degradation and pigment-binder interactions. The protein content of homemade paint samples was extracted using different methods and analysed to select the best extraction strategy based on the number of peptides that were identified. A large dataset of 4 binders (animal glue, egg yolk, egg white and casein), mixed with 10 common pigments with different chemical properties was used to study the influence of pigments on the extraction method. Analytical characteristics of the selected method were determined. Finally the method was applied to historic paint samples. The results were compared with those obtained by traditional amino acid analysis methods

PiTRAQ, a strategy to simultaneously correlate protein expression and phosphorylation stoichiometry between different samples: evaluation on different mass spectrometers

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iTRAQ as a method for optimization : enhancing peptide recovery after gel fractionation

At the dawn of a new era in label-free quantitation on high-resolution MS instruments, classical methods such as iTRAQ continue to provide very useful insights in comparative proteomics. The potential to multiplex samples makes this reporter-based labeling technique highly suited for method optimization as demonstrated here by a set of standard series. Instead of studying ratios of annotated proteins, we propose an alternative method, based on the analysis of the average reporter ratios of all the spectra from a sample or a large distinct subset herein. This strategy circumvents the bias, associated with the annotation and iTRAQ quantitation, leading to increased adequacy in measuring yield differences between workflows. As gel electrophoresis prior to MS analysis is highly beneficial, for example, as a fractionation step, the approach was applied to evaluate the influence of several parameters of the established in-gel digestion protocol. We quantified the negative effect of SYPRO Ruby staining and the positive effect of gel fixation prior to digestion on peptide yield. Finally, we emphasize the benefits of adding CaCl2 and ACN to a tryptic in-gel digest, resulting in an up to tenfold enhanced peptide recovery and fewer trypsin missed cleavages

Minimizing technical variation during sample preparation prior to label-free quantitative mass spectrometry

Sample preparation is the crucial starting point to obtain high-quality mass spectrometry data and can be divided into two main steps in a bottom-up proteomics approach: cell/tissue lysis with or without detergents and a(n) (in-solution) digest comprising denaturation, reduction, alkylation, and digesting of the proteins. Here, some important considerations, among others, are that the reagents used for sample preparation can inhibit the digestion enzyme (e.g., 0.1% sodium dodecyl sulfate [SDS] and 0.5 M guanidine HCl), give rise to ion suppression (e.g., polyethylene glycol [PEG]), be incompatible with liquid chromatography tandem mass spectrometry (LC MS/MS) (e.g., SDS), and can induce additional modifications (e.g., urea). Taken together, all of these irreproducible effects are gradually becoming a problem when label-free quantitation of the samples is envisioned such as during the increasingly popular high-definition mass spectrometry (HDMSE) and sequential window acquisition of all theoretical fragment ion spectra (SWATH) data-independent acquisition strategies. Here, we describe the detailed validation of a reproducible method with sufficient protein yield for sample preparation without any known LC MS/MS interfering substances by using 1% sodium deoxycholate (SDC) during both cell lysis and in-solution digest