Recovery Of Peptides Research Articles

The Choice of HLA‐Associated Peptide Enrichment and Purification Strategy Affects Peptide Yields and Creates a Bias in Detected Sequence Repertoire

Understanding the most appropriate workflow for biochemical human leukocyte antigen (HLA)-associated peptide enrichment prior to ligand sequencing is essential to achieve optimal sensitivity in immunopeptidomics experiments. The use of different detergents for HLA solubilization as well as complementary workflows to separate HLA-bound peptides from HLA protein complex components after their immunoprecipitation including HPLC, C18 cartridge, and 5 kDa filter are described. It is observed that all solubilization approaches tested led to similar peptide ligand identification rates; however, a higher number of peptides are identified in samples lysed with CHAPS compared with other methods. The HPLC method is superior in terms of HLA-I peptide recovery compared with 5 kDa filter and C18 cartridge peptide purification methods. Most importantly, it is observed that both the choice of detergent and peptide purification strategy creates a significant bias for the identified peptide sequences, and that allele-specific peptide repertoires are affected depending on the workflow of choice. The results highlight the importance of employing a suitable strategy for HLA peptide enrichment and that the obtained peptide repertoires do not necessarily reflect the true distributions of peptide sequences in the sample.

Recovery of antioxidant and antimicrobial peptides through the reutilization of Nile perch wastewater by biodegradation using two Bacillus species

Nile perch wastewater was biodegraded using two Bacillus species to recover bioactive substances to enhance its reutilization value. The two Bacillus species successfully produced low-molecular-weight substances with a 47.8% degree of hydrolysis. The antioxidant activities of the Nile perch wastewater increased as the biodegradation proceeded, and the culture supernatant exhibited the highest DPPH (80.1%), ABTS (93.1%) and Fe2+ chelating (88.5%) antioxidant activities at 60 h. The antioxidant potential of the biodegraded Nile perch wastewater was found to be higher than those of other fish hydrolysates. Moreover, the biodegraded Nile perch wastewater exhibited effective antimicrobial activity against Vibrio vulnificus, exhibiting a minimal inhibitory concentration of 585 μg mL−1. Two-dimensional thin layer chromatography analysis revealed the specific amino acids responsible for the antioxidant activity, and molecular-weight cut-off ultrafiltration revealed that the <2-kDa fraction exhibited the highest antioxidant activity with the lowest IC50 values (0.43 and 0.22 mg mL−1 for DPPH and ABTS antioxidant activities, respectively). This is the first report of the reutilization of Nile perch wastewater as a natural antioxidant and antimicrobial ingredient for nutraceuticals.

Peptide Selection for Accurate Targeted Protein Quantification via a Dimethylation High-Resolution Mass Spectrum Strategy with a Peptide Release Kinetic Model.

A crucial step in accurate targeted protein quantification using targeted proteomics is to determine optimal proteotypic peptides representing targeted proteins. In this study, a workflow of peptide selection to determine proteotypic peptides using a dimethylation high-resolution mass spectrum strategy with a peptide release kinetic model was investigated and applied in peptide selection of bovine serum albumin. After specificity, digestibility, recovery, and stability evaluation of tryptic peptides in bovine serum albumin, the optimal proteotypic peptide was selected as LVNELTEFAK. The quantification method using LVNELTEFAK gave a linear range of 1–100 ppm with the coefficient greater than 0.9990, and the detection limit of bovine serum albumin in milk was 0.78 mg/kg. Compared with the proteotypic peptides selected by Skyline, the method showed a better performance in method validation. The workflow exhibited high comprehensiveness and efficiency in peptide selection, facilitating accurate targeted protein quantification in the food matrix, which lack protein standards.

Improved integrative analysis of the thiol redox proteome using filter-aided sample preparation

Changes in the oxidation state of protein Cys residues are involved in cell signalling and play a key role in a variety of pathophysiological states. We had previously developed GELSILOX, an in-gel method that enables the large-scale, parallel analysis of dynamic alterations to the redox state of Cys sites and protein abundance changes. Here we present FASILOX, a further development of the GELSILOX approach featuring: i) significantly increased peptide recovery, ii) enhanced sensitivity for the detection of Cys oxidative alterations, and iii) streamlined workflow that results in shortened assay duration. In mitochondria isolated from the adipose tissue of obese, diabetic patients, FASILOX revealed a sexually dimorphic trait of Cys oxidation involving mainly mitochondrial oxidative phosphorylation complexes. These results provide the first evidence for a decreased efficiency in the antioxidant response of men as compared to women.

Ion-Pairing with Triethylammonium Acetate Improves Solid-Phase Extraction of ADP-Ribosylated Peptides.

ADP-ribosylation refers to the post-translational modification of protein substrates with monomers or polymers of the small molecule ADP-ribose. ADP-ribosylation is enzymatically regulated and plays roles in cellular processes including DNA repair, nucleic acid metabolism, cell death, cellular stress responses, and antiviral immunity. Recent advances in the field of ADP-ribosylation have led to the development of proteomics approaches to enrich and identify endogenous ADP-ribosylated peptides by liquid chromatography tandem mass spectrometry (LC-MS/MS). A number of these methods rely on reverse-phase solid-phase extraction as a critical step in preparing cellular peptides for further enrichment steps in proteomics workflows. The anionic ion-pairing reagent trifluoroacetic acid (TFA) is typically used during reverse-phase solid-phase extraction to promote retention of tryptic peptides. Here we report that TFA and other carboxylate ion-pairing reagents are inefficient for reverse-phase solid-phase extraction of ADP-ribosylated peptides. Substitution of TFA with cationic ion-pairing reagents, such as triethylammonium acetate (TEAA), improves recovery of ADP-ribosylated peptides. We further demonstrate that substitution of TFA with TEAA in a proteomics workflow specific for identifying ADP-ribosylated peptides increases identification rates of ADP-ribosylated peptides by LC-MS/MS.

How Molecular Weight Cut-Offs and Physicochemical Properties of Polyether Sulfone Membranes Affect Peptide Migration and Selectivity during Electrodialysis with Filtration Membranes.

Filtration membranes (FMs) are an integral part of electrodialysis with filtration membranes (EDFM), a green and promising technology for bioactive peptide fractionation. Therefore, it is paramount to understand how physicochemical properties of FMs impact global and selective peptide migration to anionic (A−RC) and cationic (C+RC) peptide recovery compartments during their simultaneous separation by EDFM. In this context, six polyether sulfone (PES) membranes with molecular weight cut-offs (MWCO) of 5, 10, 20, 50, 100 and 300 kDa were characterized and used during EDFM to separate peptides from a complex whey protein hydrolysate. Surface charge, roughness, thickness and surface/pores nature of studied PES membranes were similar with small differences in conductivity, porosity and pore size distribution. Interestingly, global peptides migration to both recovery compartments increased linearly as a function of MWCO. However, peptide selectivity changed according to the recovery compartments and/or the peptide’s charge and MW with an increase in MWCO of FMs. Indeed, in A−RC, the relative abundance (RA) of peptides having low negative charge and MW (IDALNENK and VLVLDTDYK) decreased (45% to 19%) with an increase in MWCO, while the opposite for peptides having high negative charge and MW (TPEVDDEALEK, TPEVDDEALEKFDK & VYVEELKPTPEGDLEILLQK) (increased from 16% to 43%). Concurrently, in C+RC, regardless of MWCO used, the highest RA was observed for peptides having low positive charge and MW (IPAVFK & ALPMHIR). It was the first time that the significant impact of charge, MWCO and pore size distribution of PES membranes on a wide range of MWCO was demonstrated on EDFM performances.

Rapid Identification of Protein-Protein Interactions in Plants.

Enzyme-enzyme interactions can be discovered by affinity purification mass spectrometry (AP-MS) under in vivo conditions. Tagged enzymes can either be transiently transformed into plant leaves or stably transformed into plant cells prior to AP-MS. The success of AP-MS depends on the levels and stability of the bait protein, the stability of the protein-protein interactions, and the efficiency of trypsin digestion and recovery of tryptic peptides for MS analysis. Unlike in-gel-digestion AP-MS, in which the gel is cut into pieces for several independent trypsin digestions, we uses a proteomics-based in-solution digestion method to directly digest the proteins on the beads following affinity purification. Thus, a single replicate within an AP-MS experiment constitutes a single sample for LC-MS measurement. In subsequent data analysis, normalized signal intensities can be processed to determine fold-change abundance (FC-A) scores by use of the SAINT algorithm embedded within the CRAPome software. Following analysis of co-sublocalization of "bait" and "prey," we suggest considering only the protein pairs for which the intensities were more than 2% compared with the bait, corresponding to FC-A values of at least four within-biological replicates, which we recommend as minimum. If the procedure is faithfully followed, experimental assessment of enzyme-enzyme interactions can be carried out in Arabidopsis within 3 weeks (transient expression) or 5 weeks (stable expression). © 2019 The Authors. Basic Protocol 1: Gene cloning to the destination vectors Alternate Protocol: In-Fusion or Gibson gene cloning protocol Basic Protocol 2: Transformation of baits into the plant cell culture or plant leaf Basic Protocol 3: Affinity purification of protein complexes Basic Protocol 4: On-bead trypsin/LysC digestion and C18 column peptide desalting and concentration Basic Protocol 5: Data analysis and quality control.

Sodium dodecyl sulfate free gel electrophoresis/electroelution sorting for peptide fractionation.

Shotgun proteomics based on peptide fractionation by using liquid chromatography has become the common procedure for proteomic studies, although in the very beginning of the field, protein separation by using electrophoresis was the main tool. Nonetheless, during the last two decades, the electrophoretic techniques for peptide mixtures fractionation have evolved as a result of relevant technological improvements. We also proposed the combination of sodium dodecyl sulfate polyacrylamide gel electrophoresis for protein fractionation and sodium dodecyl sulfate free polyacrylamide gel electrophoresis for peptide separation as a novel procedure for proteomic studies. Here, we present an optimized device for sodium dodecyl sulfate free polyacrylamide gel electrophoresis improving peptide recoveries respect to the established electrophoretic technique off gel electrophoresis meanwhile conserving the excellent resolution described for the former technique in slab gel based systems. The device simultaneously allows the separation and the collection of fractionated peptides in solution.

Intracellular Proteomic Analysis of Streptomyces sp. MC1 When Exposed to Cr(VI) by Gel-Based and Gel-Free Methods.

The actinobacterium Streptomyces sp. MC1 has previously shown the capacity to resist and remove Cr(VI) from liquid culture media. The aim of this work is to analyze the differential expression pattern of intracellular proteins when Streptomyces sp. MC1 is exposed to Cr(VI) in order to explain the molecular mechanisms of resistance that this microorganism possesses. For this purpose, 2D-PAGE and shotgun proteomic analyses (2D-nanoUPLC-ESI-MS/MS) were applied. The presence of Cr(VI) induced the expression of proteins involved in molecular biosynthesis and energy generation, chaperones with a key role in the repair of misfolded proteins and stress response, transcription proteins, proteins of importance in the DNA supercoiling, repair and replication, and dehydrogenases involved in oxidation-reduction processes. These dehydrogenases can be associated with the reduction of Cr(VI) to Cr(III). The results of this study show that proteins from the groups mentioned before are important to face the stress caused by the Cr(VI) presence and help the microorganism to counteract the toxicity of the metal. The use of two proteomic approaches resulted in a larger number of peptides identified, which is also transduced in a significant number of protein ID. This decreased the potential complexity of the sample because of the protein dynamic range, as well as increased the recovery of peptides from the gel after digestion.

Valorisation of fish discards assisted by enzymatic hydrolysis and microbial bioconversion: Lab and pilot plant studies and preliminary sustainability evaluation

The new EU fishing policies (Landing Obligation) are aimed at preventing the elimination of fishing discards overboard. These new biomasses that have to be landed from 2019 force to stablish valorisation protocols since, in most cases, they cannot be used directly for human consumption. In this context, the aim of this work was to develop an integral process based on enzyme proteolysis that permitted jointly the production and recovery of fish protein hydrolysates (FPHs), oils, bioactive peptides and fish peptones. This procedure was initially applied to ten fish discards to lab scale. FPHs of high quality in terms of soluble protein and amino acid contents, digestibility and bioactivities were obtained. The growth and metabolites productions by Pediococcus acidilactici on peptones from FPHs was also evaluated with excellent results. Pilot plant trials confirmed the results of FPHs production obtained at lab scale. Finally, a comparison with the nowadays most common use of fish biomass (fish meal production) has been made as a preliminary sustainability assessment of the proposed FPHs valorisation chain.

Proteins and Co-products from Seafood Processing Discards: Their Recovery, Functional Properties and Applications

Commercial processing of seafood results in enormous amounts of solid discards, offal or by-products. These discards, on dry weight basis, contain up to 60% proteins, consisting mostly of myofibrillar proteins, collagen, enzymes, and also soluble nitrogenous compounds. In view of their nutritional and functional values, there is a need for recovery of these proteins from the discards, which will help better utilization of harvested and farmed seafood besides reducing seafood-associated environmental problems. Iso-electric pH solubilization precipitation is a plausible method for the recovery of proteins from fishery discards. The recovered proteins are comparable with conventional surimi in gel forming and other functional properties. Other approaches for protein recovery include mechanical deboning of fish frames, development of weak acid-induced gels, protein dispersions, and treatment of the discards by proteolytic enzymes. This article discusses recovery of proteins, enzymes, protein hydrolyzates and peptides from seafood processing discards, their nutritional, bioactive and functional properties, as well as their food and nutraceutical applications.

Sequential hydrolysis of spent brewer's yeast improved its physico-chemical characteristics and antioxidant properties: A strategy to transform waste into added-value biomolecules

Abstract The spent brewer's yeast (SBY) is a promising raw material due to its high content of proteins, but cells must be disrupted to release yeast compounds. Our work aimed to study the rupturing of SBY cell wall comparing conventional methods (autolysis and mechanical rupture) with enzymatic hydrolysis using proteolytic enzymes (Brauzyn®, Alcalase™, Protamex™ and Flavourzyme™). The susceptibility to rupture of different SBY by-products (repitched and non-repitched) and the effect of sequential enzymatic hydrolysis after rupture were also investigated. The hydrolysate produced at pH 5.5, 100% substrate concentration, 10% enzyme/substrate ratio and 60 °C resulted in maximized yield and enhanced antioxidant properties. Yeast compounds were more efficiently released after enzymatic hydrolysis, resulting in increases of 50% in crude protein, 83% in protein recovery and 63% in antioxidant properties. Repitched yeast took 3.5 longer to achieve the same degree of hydrolysis of non-repitched SBY. Sequential hydrolysis using Brauzyn® and Alcalase™ resulted in maximum solid recovery and antioxidant properties. An effective approach for the recovery of proteins and peptides of SBY while reducing environmental impact of beer production was presented. Additionally, we demonstrated that a process to reuse SBY must contemplate yeasts differences and their susceptibility to breakdown to be successfully implemented.

Peptidomic and Bioinformatic Analysis to Evaluate the Application of Peptides from Latex of Hevea brasiliensis Clone BPM24

Peptidomic analysis coupled with mass spectrometry identification was employed to investigate the constitutively expressed peptides in latex serum Phytophthora-resistant rubber tree (Hevea brasiliensis), BPM24. The utilization of proteins and peptides as a strategy to study new methods can lead to a therapeutic application. However, a high-efficiency method to extract peptides from latex serum is not well established. After optimization of latex serum isolation from natural rubber latex, using of lysis solution for peptide extraction exhibited superior serum peptide recovery than using other chemical reagents. Ultrafiltration followed by solid-phase extraction was selected to achieve high extraction yield of low molecular weight proteins and peptides. Mass spectrometric and bioinformatics analysis characterized and predicted the identified protein and peptide contents. All latex serum peptide fractions from C18 reversed-phase chromatography possessed effectiveness of antifungal activity against three tested plant fungal including A. brassicicola, C. lunata and M. grisea with different MIC in the range 250-2,000 μg/ml from different peptide fraction. Moreover, Etotal (E1-E5 pooled together) from lysis solution extraction was found to able to inhibit three tested plant fungal at MIC 500 μg/ml. Moreover, B. cereus and E. faecium were the sensitive gram-positive bacteria with MIC in the range 250-4,000 μg/ml. However, all gram-negative bacteria were not affected by 4,000 μg/ml. Among these tested pathogens, plant fungal was quite sensitive to our peptide product. Etotal from lysis solution extraction will be the best candidate for using further in antifungal testing of other plant fungal both in vitro and in vivo. The further investigation and application will lead to possible use of latex serum as a great biotechnological resource to develop peptide –based product for the treatment of plant infection.

Prolonged Suppressive Effects of Periodontitis on Salivary TFF3 Production.

Objective As a follow-up to our previous study that demonstrated decreased salivary trefoil factor family 3 (TFF3) peptide levels in chronic periodontitis patients, this current study aimed to observe the effects of nonsurgical periodontal treatment on salivary TFF3 peptides in patients with periodontal diseases. Materials and Methods Eighty-seven volunteers that comprised of 30 individuals with healthy periodontium, 31 with gingivitis, and 26 with chronic periodontitis were considered for the study. Prior to periodontal treatment, a general periodontal examination was performed along with collection of saliva samples from each volunteer. Nonsurgical periodontal treatments were provided to patients with gingivitis and periodontitis. Two weeks post-treatment, saliva samples were recollected, and the periodontal status was re-evaluated. Salivary TFF3 concentrations were measured by enzyme-linked immunosorbent assay. Statistical Analysis Mann–Whitney U test was used when the investigated data were not normally distributed. Chi-squared test was used when dealing with categorical data. Kruskal–Wallis test with post-hoc corrections was used to compare data among the three investigated groups. Two-tailed p < 0.05 was considered as statistically significant. Results Prior to the periodontal treatment, salivary TFF3 concentrations in patients with gingivitis and periodontitis were significantly lower than those with healthy periodontium. Two weeks post-treatment, increased levels of salivary TFF3 were observed in patients with gingivitis, whereas the concentrations decreased in patients with chronic periodontitis. Conclusion This study demonstrated the effects of periodontal disease on the production of salivary TFF3 peptides. Interestingly, nonsurgical periodontal treatment also affected the recovery of salivary TFF3 peptides but varied in their outcomes between gingivitis and periodontitis patients.

How Charge and Triple Size-Selective Membrane Separation of Peptides from Salmon Protein Hydrolysate Orientate their Biological Response on Glucose Uptake.

The valorization of by-products from natural organic sources is an international priority to respond to environmental and economic challenges. In this context, electrodialysis with filtration membrane (EDFM), a green and ultra-selective process, was used to separate peptides from salmon frame protein hydrolysate. For the first time, the simultaneous separation of peptides by three ultrafiltration membranes of different molecular-weight exclusion limits (50, 20, and 5 kDa) stacked in an electrodialysis system, allowed for the generation of specific cationic and anionic fractions with different molecular weight profiles and bioactivity responses. Significant decreases in peptide recovery, yield, and molecular weight (MW) range were observed in the recovery compartments depending on whether peptides had to cross one, two, or three ultrafiltration membranes. Moreover, the Cationic Recovery Compartment 1 fraction demonstrated the highest increase (42%) in glucose uptake on L6 muscle cells. While, in the anionic configuration, both Anionic Recovery Compartment 2 and Anionic Recovery Compartment 3 fractions presented a glucose uptake response in basal condition similar to the insulin control. Furthermore, Cationic Recovery Compartment 3 was found to contain inhibitory peptides. Finally, LC-MS analyses of the bioassay-guided bioactive fractions allowed us to identify 11 peptides from salmon by-products that are potentially responsible for the glucose uptake improvement.

Microanalytical Mass Spectrometry Detects RAS Metabolites in Small Identified Mouse Brain Samples

BackgroundThere is increasing evidence for cardiovascular disease (CVD) development in post‐traumatic stress disorder (PTSD). The renin‐angiotensin system (RAS) critical for cardiovascular homeostasis, has been identified as potential therapeutic target and link to comorbid PTSD‐CVD development. However, the mechanisms are unclear. To better understand how the RAS contributes to CVD risk in PTSD, measurements of the RAS metabolites, the angiotensin peptides, in targeted brain areas are necessary. The low concentrations of angiotensin peptides in the brain however, challenges modern analytical approaches. This challenge is further compounded when studying small identified areas such as brain tissue punches. As a response to this challenge, we developed a microanalytical mass spectrometry approach to enable the detection of angiotensin peptides from brain tissue punches.MethodsUsing 6 angiotensin‐like peptide standards, we reconfigured a laboratory‐built capillary electrophoresis electrospray ionization platform (CE‐ESI) platform and a high‐resolution mass spectrometer (HRMS) for targeted peptide detection, essentially serving as a highly confident peptide assay. We benchmarked our approach to nano‐liquid chromatography (LC) ESI‐HRMS, the traditional technology for peptide analysis in HRMS.ResultsWe first developed a targeted approach on a quadrupole‐orbitrap mass spectrometer for the detection and quantification of the 6 selected angiotensin‐like peptides. Comparison of our CE‐ESI‐HRMS approach to the traditional nanoLC‐ESI‐HRMS revealed comparable peptide signals despite a 5‐fold reduction in sample loading. This demonstrated that our platform was suited for the measurement of angiotensin peptides from small sample volumes. The lower‐limit of quantification was established in the pM–nM range for all the selected RAS metabolites. After revising the extraction method to enhance peptide recovery, we applied our methodology to measure different levels of RAS metabolites in brain punches from the subfornical organ (SFO) and the paraventricular nucleus of the hypothalamus (PVN) of control and water deprived (WD) mice. Differences in the level of all RAS metabolites in the SFO concurred with the effect of WD in increasing angiotensin levels. On the other hand, levels for most of the RAS metabolites in the PVN did not change noticeably.ConclusionsThese results demonstrate that our CE‐ESI‐HRMS based analytical approach enabled the sensitive detection of RAS metabolites in small brain tissue samples, raising a potential to help study the chemistry of the brain RAS during PTSD and CVD.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reliable LC-MS Multiattribute Method for Biotherapeutics by Run-Time Response Calibration.

A major challenge of a mass-spectrometry-based quantitative multiattribute method (MAM) for biotherapeutics is its high variability between instruments. For reproducible attribute measurements, not only is a similar instrument model required, but the instruments must also be tuned to the same condition. This poses great long-term challenges, considering the rapid development of new instrumentations. In addition, differences in digestion efficiency, peptide recovery, and artificial modifications during sample preparation also contribute to variability between laboratories. To overcome these challenges, new mathematical methods are developed to calculate the attribute abundance in the sample, using the reference standard (RS) material as calibrant. Most quality attributes in the RS remain constant throughout the life of the standard, and therefore, the RS can serve as a calibrant to correct for the difference between instruments or sample preparation procedures. Because RS data are usually collected in a MAM assay, no additional work is required from the analyst. Data from a large number of attributes demonstrated that these methodologies greatly reduced instrument-to-instrument and sample preparation variabilities. With these methodologies, a consistent instrument model and sample preparation procedure is no longer a requirement. As a result, changes in digestion procedure and advances in instrumentations will not significantly affect the assay result.

Vitamin D for the Immune System in Cystic Fibrosis (DISC): a double-blind, multicenter, randomized, placebo-controlled clinical trial

Patients with cystic fibrosis (CF) have increased risk of vitamin D deficiency owing to fat malabsorption and other factors. Vitamin D deficiency has been associated with increased risk of pulmonary exacerbations of CF. The primary objective of this study was to examine the impact of a single high-dose bolus of vitamin D3 followed by maintenance treatment given to adults with CF during an acute pulmonary exacerbation on future recurrence of pulmonary exacerbations. This was a multicenter, double-blind, placebo-controlled, intent-to-treat clinical trial. Subjects with CF were randomly assigned to oral vitamin D3 given as a single dose of 250,000 International Units (IU) or to placebo within 72 h of hospital admission for an acute pulmonary exacerbation, followed by 50,000 IU of vitamin D3 or an identically matched placebo pill taken orally every other week starting at 3 mo after random assignment. The primary outcome was the composite endpoint of the time to next pulmonary exacerbation or death within 1 y. The secondary outcomes included circulating concentrations of the antimicrobial peptide cathelicidin and recovery of lung function as assessed by the percentage of predicted forced expiratory volume in 1 s (FEV1%). A total of 91 subjects were enrolled in the study. There were no differences between the vitamin D3 and placebo groups in time to next pulmonary exacerbation or death at 1 y. In addition, there were no differences in serial recovery of lung function after pulmonary exacerbation by FEV1% or in serial concentrations of plasma cathelicidin. Vitamin D3 initially given at the time of pulmonary exacerbation of CF did not alter the time to the next pulmonary exacerbation, 12-mo mortality, serial lung function, or serial plasma cathelicidin concentrations. This trial was registered at clinicaltrials.gov as NCT01426256.

SP2: Rapid and Automatable Contaminant Removal from Peptide Samples for Proteomic Analyses.

Peptide cleanup is essential for the removal of contaminating substances that may be introduced during sample preparation steps in bottom-up proteomic workflows. Recent studies have described benefits of carboxylate-modified paramagnetic particles over traditional reversed-phase methods for detergent and polymer removal, but challenges with reproducibility have limited the widespread implementation of this approach among laboratories. To overcome these challenges, the current study systematically evaluated key experimental parameters regarding the use of carboxylate-modified paramagnetic particles and determined those that are critical for maximum performance and peptide recovery and those for which the protocol is tolerant to deviation. These results supported the development of a detailed, easy-to-use standard operating protocol, termed SP2, which can be applied to remove detergents and polymers from peptide samples while concentrating the sample in solvent that is directly compatible with typical LC-MS workflows. We demonstrate that SP2 can be applied to phosphopeptides and glycopeptides and that the approach is compatible with robotic liquid handling for automated sample processing. Altogether, the results of this study and accompanying detailed operating protocols for both manual and automated processing are expected to facilitate reproducible implementation of SP2 for various proteomics applications and will especially benefit core or shared resource facilities where unknown or unexpected contaminants may be particularly problematic.

Detection of Six Commercially Processed Soy Ingredients in an Incurred Food Matrix Using Parallel Reaction Monitoring

Soybeans are one of the major allergenic foods in many countries. Soybeans are commonly processed into different types of soy ingredients to achieve the desired properties. The processing, however, may affect the protein profiles and protein structure, thus affecting the detection of soy proteins. Mass spectrometry (MS) is a potential alternative to the traditional immunoassays for the detection of soy-derived ingredients in foods. This study aims to develop a liquid chromatography-tandem MS method that uniformly detects different types of soy-derived ingredients. Target peptides applicable to the detection of six commercial soy ingredients were identified based on the results of MS label-free quantification and a set of selection criteria. The results indicated that soy ingredient processing can result in different protein profiles. A total of six soy ingredients were then individually incurred into cookie matrices at different levels. Sample preparation methods were optimized, and a distinct improvement in peptide performance was observed after optimization. Cookies and dough incurred with different soy ingredients at 100 ppm total soy protein showed a similar level of peptide recovery (90% mean signal relative to unroasted soy flour), demonstrating the ability of the MS method to detect processed soy ingredients in a uniform manner.