An interlaboratory capillary zone electrophoresis-UV study of various monoclonal antibodies, instruments, and ε-aminocaproic acid lots.

Capillary zone electrophoresis ultraviolet (CZE-UV) has become increasingly popular for the charge heterogeneity determination of mAbs and vaccines. The ε-aminocaproic acid (eACA) CZE-UV method has been used as a rapid platform method. However, in the last years, several issues have been observed, for example, loss in electrophoretic resolution or baseline drifts. Evaluating the role of eACA on the reported issues, various laboratories were requested to provide their routinely used eACA CZE-UV methods, and background electrolyte compositions. Although every laboratory claimed to use the He et al. eACA CZE-UV method, most methods actually deviate from He's. Subsequently, a detailed interlaboratory study was designed wherein two commercially available mAbs (Waters' Mass Check Standard mAb [pI 7] and NISTmAb [pI 9]) were provided to each laboratory, along with two detailed eACA CZE-UV protocols for a short-end, high-speed, and a long-end, high-resolution method. Ten laboratories participated each using their own instruments, and commodities, showing excellence method performance (relative standard deviations [RSDs] of percent time-corrected main peak areas from 0.2% to 1.9%, and RSDs of migration times from 0.7% to 1.8% [n = 50 per laboratory], analysis times in some cases as short as 2.5 min). This study clarified that eACA is not the main reason for the abovementioned variations.

Capillary Electrophoresis-Mass Spectrometry at Trial by Metabo-Ring: Effective Electrophoretic Mobility for Reproducible and Robust Compound Annotation.

Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (µeff) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma, and urine samples spiked with the same metabolite mixture were used and distributed for analysis by all laboratories. The µeff was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning, and rinsing procedure, etc.) were left to the discretion of the contributing laboratories. The results revealed that the reproducibility of the µeff for 20 out of the 21 model compounds was below 3.1% vs 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the 13 laboratories. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics.

Extensive mass spectrometry-based analysis of the fission yeast proteome: the Schizosaccharomyces pombe PeptideAtlas.

We report a high quality and system-wide proteome catalogue covering 71% (3,542 proteins) of the predicted genes of fission yeast, Schizosaccharomyces pombe, presenting the largest protein dataset to date for this important model organism. We obtained this high proteome and peptide (11.4 peptides/protein) coverage by a combination of extensive sample fractionation, high resolution Orbitrap mass spectrometry, and combined database searching using the iProphet software as part of the Trans-Proteomics Pipeline. All raw and processed data are made accessible in the S. pombe PeptideAtlas. The identified proteins showed no biases in functional properties and allowed global estimation of protein abundances. The high coverage of the PeptideAtlas allowed correlation with transcriptomic data in a system-wide manner indicating that post-transcriptional processes control the levels of at least half of all identified proteins. Interestingly, the correlation was not equally tight for all functional categories ranging from r(s) >0.80 for proteins involved in translation to r(s) <0.45 for signal transduction proteins. Moreover, many proteins involved in DNA damage repair could not be detected in the PeptideAtlas despite their high mRNA levels, strengthening the translation-on-demand hypothesis for members of this protein class. In summary, the extensive and publicly available S. pombe PeptideAtlas together with the generated proteotypic peptide spectral library will be a useful resource for future targeted, in-depth, and quantitative proteomic studies on this microorganism.

Compliance and regulatory considerations for the implementation of the multi-attribute-method by mass spectrometry in a quality control laboratory.

Multi-attribute methods employing mass spectrometry are applied throughout the biopharmaceutical industry for product and process characterization purposes but are not yet widely accepted as a method for batch release and stability testing under the good manufacturing practice (GMP) regime, due to limited experience and level of comfort with the technical, compliance and regulatory aspects of its implementation at quality control (QC) laboratories. This article is the second part of a two-tiered publication aiming at providing guidance for implementation of the multi-attribute method by peptide mapping liquid chromatography mass spectrometry (MAM) in a QC laboratory. The first part [1] focuses on technical considerations, while this second part provides considerations related to GMP compliance and regulatory aspects. This publication has been prepared by a group of industry experts representing 14 globally acting major biotechnology companies under the umbrella of the European Federation of Pharmaceutical Industries and Associations (EFPIA) Manufacturing & Quality Expert Group (MQEG).

Technical considerations for the implementation of the multi-attribute-method by mass spectrometry in a quality control laboratory.

Multi-attribute methods employing mass spectrometry are applied throughout the biopharmaceutical industry for product and process characterization purposes but are not yet widely accepted as a method for batch release and stability testing under good manufacturing practice (GMP) due to limited experience and level of comfort with the technical, compliance and regulatory aspects of its implementation at quality control (QC) laboratories. Here, current literature related to the development and application of the multi-attribute method by peptide mapping liquid chromatography mass spectrometry (MAM) is compiled with the aim of providing guidance for the implementation of MAM in a QC laboratory. This article, focusing on technical considerations, is the first part of a two-tiered publication, whereby the second part will focus on GMP compliance and regulatory aspects. This publication has been prepared by a group of industry experts representing 14 globally acting major biotechnology companies under the umbrella of the European Federation of Pharmaceutical Industries and Associations (EFPIA) Manufacturing & Quality Expert Group (MQEG).

Expanding the zebrafish embryo proteome using multiple fractionation approaches and tandem mass spectrometry.

The proteome of zebrafish, Danio rerio, embryos has not been studied in great detail mainly due to the presence of high abundance yolk proteins in embryos. Here we report the highest number of the zebrafish embryo proteins identified so far to our knowledge, through a combination of a protein-level fractionation approach (1D SDS-PAGE) and two different peptide-level fractionation approaches (IEF and strong anion exchange (SAX)) of deyolked zebrafish embryos followed by LC-MS/MS. We detected 5267 proteins in total of which 3464 proteins were identified with at least two peptides (less than 1% peptide false discovery rate). The analysis of proteome coverage from each method showed that 56% of detected proteins were common to all approaches and 95% of the detected proteome was obtained from 1D SDS-PAGE approach alone. Bioinformatics analysis of the detected proteome demonstrated that nucleocytoplasmic transport (biological process) and ribosomal proteins (cellular component) were the most over-represented proteins, whereas cell-cell signaling (biological process) and extracellular space proteins (cellular component) were the most under-represented proteins in the identified proteome.

Preventing arginine-to-proline conversion in a cell-line-independent manner during cell cultivation under stable isotope labeling by amino acids in cell culture (SILAC) conditions.

Quantitative proteomics has increasingly gained impact in life science research as a tool to describe changes in protein expression between different cellular states. Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful technique for relative quantification of proteins. However, the accuracy of quantification is impaired by the metabolic conversion of arginine to proline resulting in additional heavy labeled proline peptide satellites. Here we reinvestigated the addition of unlabeled proline during cell cultivation under SILAC conditions considering several thousand peptides and demonstrated that the arginine-to-proline conversion is prevented independent of the cell line used.

In-depth profiling of the peripheral blood mononuclear cells proteome for clinical blood proteomics.

Peripheral blood mononuclear cells (PBMCs) are an easy accessible cellular part of the blood organ and, along with platelets, represent the only site of active gene expression in blood. These cells undergo immunophenotypic changes in various diseases and represent a peripheral source of monitoring gene expression and posttranslational modifications relevant to many diseases. Little is known about the source of many blood proteins and we hypothesise that release from PBMCs through active and passive mechanisms may account for a substantial part of the plasma proteome. The use of state-of-the-art proteomic profiling methods in PBMCs will enable minimally invasive monitoring of disease progression or response to treatment and discovery of biomarkers. To achieve this goal, detailed mapping of the PBMC proteome using a sensitive, robust, and quantitative methodological setup is required. We have applied an indepth gel-free proteomics approach using tandem mass tags (TMT), unfractionated and SCX fractionated PBMC samples, and LC-MS/MS with various modulations. This study represents a benchmark in deciphering the PBMC proteome as we provide a deep insight by identifying 4129 proteins and 25503 peptides. The identified proteome defines the scope that enables PBMCs to be characterised as cellular major biomarker pool within the blood organ.

Quantification of pancreatic cancer proteome and phosphorylome: indicates molecular events likely contributing to cancer and activity of drug targets.

OBJECTIVE: LC-MS/MS phospho-proteomics is an essential technology to help unravel the complex molecular events that lead to and propagate cancer. We have developed a global phospho-proteomic workflow to determine activity of signaling pathways and drug targets in pancreatic cancer tissue for clinical application. METHODS: Peptides resulting from tryptic digestion of proteins extracted from frozen tissue of pancreatic ductal adenocarcinoma and background pancreas (n = 12), were labelled with tandem mass tags (TMT 8-plex), separated by strong cation exchange chromatography, then were analysed by LC-MS/MS directly or first enriched for phosphopeptides using IMAC and TiO2, prior to analysis. In-house, commercial and freeware bioinformatic platforms were used to identify relevant biological events from the complex dataset. RESULTS: Of 2,101 proteins identified, 152 demonstrated significant difference in abundance between tumor and non-tumor tissue. They included proteins that are known to be up-regulated in pancreatic cancer (e.g. Mucin-1), but the majority were new candidate markers such as HIPK1 & MLCK. Of the 6,543 unique phosphopeptides identified (6,284 unique phosphorylation sites), 635 showed significant regulation, particularly those from proteins involved in cell migration (Rho guanine nucleotide exchange factors & MRCKα) and formation of focal adhesions. Activator phosphorylation sites on FYN, AKT1, ERK2, HDAC1 and other drug targets were found to be highly modulated (≥2 fold) in different cases highlighting their predictive power. CONCLUSION: Here we provided critical information enabling us to identify the common and unique molecular events likely contributing to cancer in each case. Such information may be used to help predict more bespoke therapy suitable for an individual case.

Enhanced performance of pulsed Q collision induced dissociation-based peptide identification on a dual-pressure linear ion trap.

Pulsed Q collision induced dissociation (PQD) was introduced for isobaric tag quantification on linear ion traps to circumvent the problem of the low-mass cut-off for collision induced dissociation (CID). Unfortunately, fragmentation efficiency is compromised and PQD has found limited use for identification as well as quantification. We demonstrate that PQD has a comparable peptide identification performance to CID on dual-pressure linear ion traps, opening the potential for wider use of isobaric tag quantification on this new generation of linear ion traps.

Quantitative proteomics identify novel miR-155 target proteins.

BACKGROUND: MicroRNAs are 22 nucleotides long non-coding RNAs and exert their function either by transcriptional or translational inhibition. Although many microRNA profiles in different tissues and disease states have already been discovered, only little is known about their target proteins. The microRNA miR-155 is deregulated in many diseases, including cancer, where it might function as an oncoMir. METHODOLOGY/PRINCIPAL FINDINGS: We employed a proteomics technique called "stable isotope labelling by amino acids in cell culture" (SILAC) allowing relative quantification to reliably identify target proteins of miR-155. Using SILAC, we identified 46 putative miR-155 target proteins, some of which were previously reported. With luciferase reporter assays, CKAP5 was confirmed as a new target of miR-155. Functional annotation of miR-155 target proteins pointed to a role in cell cycle regulation. CONCLUSIONS/SIGNIFICANCE: To the best of our knowledge we have investigated for the first time miR-155 target proteins in the HEK293T cell line in large scale. In addition, by comparing our results to previously identified miR-155 target proteins in other cell lines, we provided further evidence for the cell line specificity of microRNAs.