The Impact of Commonly Used Alkylating Agents on Artifactual Peptide Modification

Iodoacetamide is by far the most commonly used agent for alkylation of cysteine during sample preparation for proteomics. An alternative, 2-chloroacetamide, has recently been suggested to reduce the alkylation of residues other than cysteine, such as the N-terminus, Asp, Glu, Lys, Ser, Thr, and Tyr. Here we show that although 2-chloroacetamide reduces the level of off-target alkylation, it exhibits a range of adverse effects. The most significant of these is methionine oxidation, which increases to a maximum of 40% of all Met-containing peptides, compared with 2–5% with iodoacetamide. Increases were also observed for mono- and dioxidized tryptophan. No additional differences between the alkylating reagents were observed for a range of other post-translational modifications and digestion parameters. The deleterious effects were observed for 2-chloroacetamide from three separate suppliers. The adverse impact of 2-chloroacetamide on methionine oxidation suggests that it is not the ideal alkylating reagent for proteomics

The Impact of Commonly Used Alkylating Agents on Artifactual Peptide Modification

Iodoacetamide is by far the most commonly used agent for alkylation of cysteine during sample preparation for proteomics. An alternative, 2-chloroacetamide, has recently been suggested to reduce the alkylation of residues other than cysteine, such as the N-terminus, Asp, Glu, Lys, Ser, Thr, and Tyr. Here we show that although 2-chloroacetamide reduces the level of off-target alkylation, it exhibits a range of adverse effects. The most significant of these is methionine oxidation, which increases to a maximum of 40% of all Met-containing peptides, compared with 2–5% with iodoacetamide. Increases were also observed for mono- and dioxidized tryptophan. No additional differences between the alkylating reagents were observed for a range of other post-translational modifications and digestion parameters. The deleterious effects were observed for 2-chloroacetamide from three separate suppliers. The adverse impact of 2-chloroacetamide on methionine oxidation suggests that it is not the ideal alkylating reagent for proteomics

FD5180, a Novel Protein Kinase Affinity Probe, and the Effect of Bead Loading on Protein Kinase Identification

The effects of compound loading on the identification of protein kinases (PKs) was examined using two previously reported sepharose-supported PK inhibitors (PKIs): bisindolylmaleimide X (<b>S1</b>) and CZC8004 (<b>S2</b>). Compound loadings of 0.1, 0.5, 2.5, 5, 10, 25, and 50% content and an ethanolamine-blocked control bead (no compound) were investigated. A 50% bead loading gave the highest level of PK identification for both <b>S1</b> and <b>S2</b>, extracting 34 and 55 PKs, respectively, from a single cell lysate. Control beads allowed overall identification of 23 PKs, which we term the kinase beadome, whereas sepharose-supported sunitinib (<b>S7</b>; 50% loading) identified 20, 11 of which were common to the control beads. The reliability of bead pull-downs was examined in duplicate experiments using two independently synthesized batches each of <b>S1</b> and <b>S2</b>. Bead <b>S1</b> showed high similarity in the absolute numbers of PKs identified across two experiments, at 40 and 35 PKs, of which 26 were common across the two batches of beads, with 14 and 9 unique PKs identified in each experiment. The <b>S2</b> beads extracted 61 and 64 PKs with 55 PKs common across the two bead batches examined. We also report on the development and use of a novel promiscuous PKI analogue, 2-[(5-chloro-2­{[4-(piperazin-1-yl)­phenyl]­amino}­pyrimidin-4-yl)­amino]-<i>N</i>-methylbenzene-sulfonamide (<b>S15</b>), which extracted 12 additional unique PKs over the two parent compounds from which it was designed, the combination of which identifies 160 unique PKs. <b>S15</b> was based on the common pyrimidine core scaffold of <b>S9</b> and <b>S10</b>. Thus, <b>S15</b> expands the utility of kinobeads by broadening the kinome coverage for bead-based pull-down. Combining the data for all beads across 90 and 180 min liquid chromatography–mass spectrometry (LC–MS)/MS analysis identified a total of 160 unique PKs

Heat ‘n Beat: A Universal High-Throughput End-to-End Proteomics Sample Processing Platform in under an Hour

Proteomic analysis by mass spectrometry of small (≤2 mg) solid tissue samples from diverse formats requires high throughput and comprehensive proteome coverage. We developed a nearly universal, rapid, and robust protocol for sample preparation, suitable for high-throughput projects that encompass most cell or tissue types. This end-to-end workflow extends from original sample to loading the mass spectrometer and is centered on a one-tube homogenization and digestion method called Heat ‘n Beat (HnB). It is applicable to most tissues, regardless of how they were fixed or embedded. Sample preparation was divided into separate challenges. The initial sample washing and final peptide cleanup steps were adapted to three tissue sources: fresh frozen (FF), optimal cutting temperature (OCT) compound embedded (FF-OCT), and formalin-fixed paraffin embedded (FFPE). Third, for core processing, tissue disruption and lysis were decreased to a 7 min heat and homogenization treatment, and reduction, alkylation, and proteolysis were optimized into a single step. The refinements produced near doubled peptide yield when compared to our earlier method ABLE delivered a consistently high digestion efficiency of 85–90%, reported by ProteinPilot, and required only 38 min for core processing in a single tube, with the total processing time being 53–63 min. The robustness of HnB was demonstrated on six organ types, a cell line, and a cancer biopsy. Its suitability for high-throughput applications was demonstrated on a set of 1171 FF-OCT human cancer biopsies, which were processed for end-to-end completion in 92 h, producing highly consistent peptide yield and quality for over 3513 MS runs