Phosphoproteomics Sample Preparation Impacts Biological Interpretation of Phosphorylation Signaling Outcomes

The influence of phosphoproteomics sample preparation methods on the biological interpretation of signaling outcome is unclear. Here, we demonstrate a strong bias in phosphorylation signaling targets uncovered by comparing the phosphoproteomes generated by two commonly used methods—strong cation exchange chromatography-based phosphoproteomics (SCXPhos) and single-run high-throughput phosphoproteomics (HighPhos). Phosphoproteomes of embryonic stem cells exposed to ionizing radiation (IR) profiled by both methods achieved equivalent coverage (around 20,000 phosphosites), whereas a combined dataset significantly increased the depth (>30,000 phosphosites). While both methods reproducibly quantified a subset of shared IR-responsive phosphosites that represent DNA damage and cell-cycle-related signaling events, most IR-responsive phosphoproteins (>82%) and phosphosites (>96%) were method-specific. Both methods uncovered unique insights into phospho-signaling mediated by single (SCXPhos) versus double/multi-site (HighPhos) phosphorylation events; particularly, each method identified a distinct set of previously unreported IR-responsive kinome/phosphatome (95% disparate) directly impacting the uncovered biology.

Antioxidant Activity from the Enzymatic Hydrolysates of Chlorella sorokiniana and Its Potential Peptides Identification in Combination with Molecular Docking Analysis

Chlorella sorokiniana is an edible microalga known for its high protein content with a balanced amino acid composition, nutritional value, beneficial health effect, and natural antioxidant. The enzymatic assays was used to extract the peptides of C. sorokiniana which was an uncommon method to test the antioxidant activity. In this research, protein of C. sorokiniana was extracted, purified, and hydrolyzed in several enzymes and kept at 37°C for 16h. Hence, enzymatic hydrolysate <3kDA was fractionated into 11 portions (C0%, C10%, to C100%) by using offline Strong Cation Exchange Chromatography (SCX) and their antioxidant activity was tested using DPPH (2.2-diphenyl-1-picrylhydrazyl) radical scavenging assay. The results indicated that C80%, contributed to the highest free DPPH scavenging on C. sorokiniana hydrolysate with the inhibition of 22.04%. Furthermore, to find the candidate peptides, this fraction was injected into LC-MS/MS for characterization of it's DPPH inhibitation. LSSATSAPS (m/z 1638,78) and AGLYGHPQTQEE (m/z 1328.59) are peptides that were identified and confirmed by LC-MS/MS. The molecular docking study was conducted to provide the binding simulation between these peptides and the ROS1 as the receptor. In conclusion, our results suggested that the aforementioned peptides were attached to ROS1 binding site and contributed to its potential antioxidant activity.

Simultaneous Monitoring of Monoclonal Antibody Variants by Strong Cation-Exchange Chromatography Hyphenated to Mass Spectrometry to Assess Quality Attributes of Rituximab-Based Biotherapeutics

Different manufacturing processes and storage conditions of biotherapeutics can lead to a significant variability in drug products arising from chemical and enzymatic post-translational modifications (PTMs), resulting in the co-existence of a plethora of proteoforms with different physicochemical properties. To unravel the heterogeneity of these proteoforms, novel approaches employing strong cation-exchange (SCX) high-performance liquid chromatography (HPLC) hyphenated to mass spectrometry (MS) using a pH gradient of volatile salts have been developed in recent years. Here, we apply an established SCX-HPLC-MS method to characterize and compare two rituximab-based biotherapeutics, the originator MabThera® and its Indian copy product Reditux™. The study assessed molecular differences between the two drug products in terms of C-terminal lysine variants, glycosylation patterns, and other basic and acidic variants. Overall, MabThera® and Reditux™ displayed differences at the molecular level. MabThera® showed a higher degree of galactosylated and sialylated glycoforms, while Reditux™ showed increased levels of oligomannose and afucosylated glycoforms. Moreover, the two drug products showed differences in terms of basic variants such as C-terminal lysine and N-terminal truncation, present in Reditux™ but not in MabThera®. This study demonstrates the capability of this fast SCX-HPLC-MS approach to compare different drug products and simultaneously assess some of their quality attributes.

Isolation of acetylated and unmodified protein N-terminal peptides by strong cation exchange chromatographic separation of TrypN-digested peptides

We developed a simple and rapid method to enrich protein N-terminal peptides, in which the protease TrypN is first employed to generate protein N-terminal peptides without Lys or Arg and internal peptides with two positive charges at their N-termini, and then the N-terminal peptides with or without N-acetylation are separated from the internal peptides by strong cation exchange chromatography according to a retention model based on the charge/orientation of peptides. This approach was applied to 20 μg of human HEK293T cell lysate proteins to profile the N-terminal proteome. On average, 1,550 acetylated and 200 unmodified protein N-terminal peptides were successfully identified in a single LC/MS/MS run with less than 3% contamination with internal peptides, even when we accepted only canonical protein N-termini registered in the Swiss-Prot database. Since this method involves only two steps, protein digestion and chromatographic separation, without the need for tedious chemical reactions, it should be useful for comprehensive profiling of protein N-termini, including proteoforms with neo-N-termini.

Isolation of protein N-terminal peptides by charge-mediated position-selective enrichment using strong cation exchange chromatography

ABSTRACTWe developed a simple and rapid method to enrich protein N-terminal peptides, in which the protease TrypN is first employed to generate protein N-terminal peptides without Lys or Arg and internal peptides with two positive charges at their N-termini, and then the N-terminal peptides are separated from the internal peptides by means of CHArge-Mediated Position-selective (CHAMP) enrichment using strong cation exchange (SCX) chromatography. This CHAMP-SCX approach was applied to 20 μg of human HEK293T cell lysate proteins to profile the N-terminal proteome. On average, 1,550 acetylated and 200 unmodified protein N-terminal peptides were successfully identified in a single LC/MS/MS run with less than 3% contamination with internal peptides, even when we accepted only canonical protein N-termini registered in the Swiss-Prot database. Since this method involves only two steps, protein digestion and chromatographic separation, without the need for tedious chemical reactions, it should be useful for comprehensive profiling of protein N-termini, including proteoforms with neo-N-termini.

A composite filter for low FDR of protein-protein interactions detected by in vivo cross-linking

In vivo chemical cross-linking combined with LCMSMS of digested extracts (in vivo CX-MS) can reveal stable and dynamic protein-protein interactions at a proteome wide-scale and at the peptide level. In vivo CX-MS requires a membrane permeable and cleavable cross-linker that enables isolation of target peptides and a fast and sensitive search engine to identify the linked peptides. Here we explore the use of the search engine pLink 2 for analysis of a previously obtained LCMSMS dataset from exponentially growing Bacillus subtilis treated in culture with the cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). Cross-linked peptide pairs were identified by pLink 2 in very short time at an overall FDR of < 5%. To also obtain a FDR < 5% for inter-protein cross-linked peptide pairs additional thresholds values were applied for matched fragment intensity and for the numbers of unambiguous y and b ions to be assigned for both composite peptides. Threshold values were based on a set of decoy sequences from yeast and human sequence databases. Also the mass- and charge-dependent retention times of target peptides purified by diagonal strong cation exchange chromatography were used as a criterion to distinguish true from false positives. After this filtering, pLink 2 identified more than 80% of previously reported protein-protein interactions. In addition the use of pLink 2 revealed interesting new inter-protein cross-linked peptide pairs, among others showing interactions between the global transcriptional repressor AbrB and elongation factor Tu and between the essential protein YlaN of unknown function and the ferric uptake repressor Fur.Abstract FigureHighlightsImproved protocol for identification of PPIs at low FDR by in vivo cross-linking with BAMGThe use of all intra-protein cross-linked peptide pairs as true positivesThe cytosolic aminopeptidase (AMPA_BACSU) interacts with the 50S ribosomal protein L17The transition state regulator AbrB interacts with elongation factor TuThe essential protein YlaN of unknown function interacts with the iron uptake repressor FurSignificanceImportant for reliable identification of PPIs by chemical cross-linking in vivo is a low FDR of non-redundant inter-protein peptide pairs. Here we describe how to recognize the presence of spurious interactions in a dataset of cross-linked peptide pairs enriched by 2D strong cation exchange chromatography and identified by LCMSMS by taking into account chromatographic behavior of cross-linked peptide pairs and protein abundance of corresponding peptides. Based on these criteria we assessed that the FDR of the fraction of non-redundant inter-protein cross-linked peptide pairs was approx. 20-25% by interrogating an entire species specific database at an overall FDR of 5% or 0.1% with a search engine that otherwise scores best in sensitivity among other search engines. We have defined a composite filter to decrease this high FDR of inter-protein cross-linked peptide pairs to only about 2%.