Label-Free Quantification Mass Spectrometry Identifies Protein Markers of Chemotherapy Response in High-Grade Serous Ovarian Cancer.

Eighty percent of ovarian cancer patients initially respond to chemotherapy, but the majority eventually experience a relapse and die from the disease with acquired chemoresistance. In addition, 20% of patients do not respond to treatment at all, as their disease is intrinsically chemotherapy resistant. Data-independent acquisition nano-flow liquid chromatography-mass spectrometry (DIA LC-MS) identified the three protein markers: gelsolin (GSN), calmodulin (CALM1), and thioredoxin (TXN), to be elevated in high-grade serous ovarian cancer (HGSOC) tissues from patients that responded to chemotherapy compared to those who did not; the differential expression of the three protein markers was confirmed by immunohistochemistry. Analysis of the online GENT2 database showed that mRNA levels of GSN, CALM1, and TXN were decreased in HGSOC compared to fallopian tube epithelium. Elevated levels of GSN and TXN mRNA expression correlated with increased overall and progression-free survival, respectively, in a Kaplan-Meier analysis of a large online repository of HGSOC patient data. Importantly, differential expression of the three protein markers was further confirmed when comparing parental OVCAR-5 cells to carboplatin-resistant OVCAR-5 cells using DIA LC-MS analysis. Our findings suggest that GSN, CALM1, and TXN may be useful biomarkers for predicting chemotherapy response and understanding the mechanisms of chemotherapy resistance. Proteomic data are available via ProteomeXchange with identifier PXD033785.

Magnetic enrichment of immuno-specific extracellular vesicles for mass spectrometry using biofilm-derived iron oxide nanowires.

Immuno-specific enrichment of extracellular vesicles (EVs) can provide important information into cellular pathways underpinning various pathologies and for non-invasive diagnostics, including mass spectrometry-based analyses. Herein, we report an optimised protocol for immuno-magnetic enrichment of specific EV subtypes and their subsequent processing with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Specifically, we conjugated placental alkaline phosphatase (PLAP) antibodies to magnetic iron oxide nanowires (NWs) derived from bacterial biofilms and demonstrated the utility of this approach by enriching placenta-specific EVs (containing PLAP) from cell culture media. We demonstrate efficient PLAP+ve EV enrichment for both NW-PLAP and Dynabeads™-PLAP, with high PLAP protein recovery (83.7 ± 8.9% and 83.2 ± 5.9%, respectively), high particle-to-protein ratio (7.5 ± 0.7 × 109 and 7.1 ± 1.2 × 109, respectively), and low non-specific binding of non-target EVs (7 ± 3.2% and 5.4 ± 2.2%, respectively). Furthermore, our optimized EV enrichment and processing approach identified 2518 and 2545 protein groups with LC-MS/MS for NW-PLAP and Dynabead™-PLAP, respectively, with excellent reproducibility (Pearson correlation 0.986 and 0.988). These findings demonstrate that naturally occurring iron oxide NWs have comparable performance to current gold standard immune-magnetic beads. The optimized immuno-specific EV enrichment for LC-MS/MS method provides a low-cost and highly-scalable yet efficient, high-throughput approach for quality EV proteomic studies.

Mass spectrometry imaging spatially identifies complex-type N-glycans as putative cartilage degradation markers in human knee osteoarthritis tissue.

N-Glycan alterations contribute to the pathophysiology and progression of various diseases. However, the involvement of N-glycans in knee osteoarthritis (KOA) progression at the tissue level, especially within articular cartilage, is still poorly understood. Thus, the aim of this study was to spatially map and identify KOA-specific N-glycans from formalin-fixed paraffin-embedded (FFPE) osteochondral tissue of the tibial plateau relative to cadaveric control (CTL) tissues. Human FFPE osteochondral tissues from end-stage KOA patients (n=3) and CTL individuals (n=3), aged >55 years old, were analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Overall, it was revealed that 22 N-glycans were found in the cartilage region of KOA and CTL tissue. Of those, 15 N-glycans were more prominent in KOA cartilage than CTL cartilage. We then compared sub-regions of KOA and CTL tissues based on the Osteoarthritis Research Society International (OARSI) histopathological grade (1 to 6), where 1 is an intact cartilage surface and 6 is cartilage surface deformation. Interestingly, three specific complex-type N-glycans, (Hex)4(HexNAc)3, (Hex)4(HexNAc)4, and (Hex)5(HexNAc)4, were found to be localized to the superficial fibrillated zone of degraded cartilage (KOA OARSI 2.5-4), compared to adjacent cartilage with less degradation (KOA OARSI 1-2) or relatively healthy cartilage (CTL OARSI 1-2). Our results demonstrate that N-glycans specific to degraded cartilage in KOA patients have been identified at the tissue level for the first time. The presence of these N-glycans could further be evaluated as potential diagnostic and prognostic biomarkers.

Chemoresistant Cancer Cell Lines Are Characterized by Migratory, Amino Acid Metabolism, Protein Catabolism and IFN1 Signalling Perturbations.

Chemoresistance remains the major barrier to effective ovarian cancer treatment. The molecular features and associated biological functions of this phenotype remain poorly understood. We developed carboplatin-resistant cell line models using OVCAR5 and CaOV3 cell lines with the aim of identifying chemoresistance-specific molecular features. Chemotaxis and CAM invasion assays revealed enhanced migratory and invasive potential in OVCAR5-resistant, compared to parental cell lines. Mass spectrometry analysis was used to analyse the metabolome and proteome of these cell lines, and was able to separate these populations based on their molecular features. It revealed signalling and metabolic perturbations in the chemoresistant cell lines. A comparison with the proteome of patient-derived primary ovarian cancer cells grown in culture showed a shared dysregulation of cytokine and type 1 interferon signalling, potentially revealing a common molecular feature of chemoresistance. A comprehensive analysis of a larger patient cohort, including advanced in vitro and in vivo models, promises to assist with better understanding the molecular mechanisms of chemoresistance and the associated enhancement of migration and invasion.

Cancer Tissue Classification Using Supervised Machine Learning Applied to MALDI Mass Spectrometry Imaging.

Matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) can determine the spatial distribution of analytes such as protein distributions in a tissue section according to their mass-to-charge ratio. Here, we explored the clinical potential of machine learning (ML) applied to MALDI MSI data for cancer diagnostic classification using tissue microarrays (TMAs) on 302 colorectal (CRC) and 257 endometrial cancer (EC)) patients. ML based on deep neural networks discriminated colorectal tumour from normal tissue with an overall accuracy of 98% in balanced cross-validation (98.2% sensitivity and 98.6% specificity). Moreover, our machine learning approach predicted the presence of lymph node metastasis (LNM) for primary tumours of EC with an accuracy of 80% (90% sensitivity and 69% specificity). Our results demonstrate the capability of MALDI MSI for complementing classic histopathological examination for cancer diagnostic applications.

In-House Packed Porous Graphitic Carbon Columns for Liquid Chromatography-Mass Spectrometry Analysis of N-Glycans.

Protein glycosylation is a common post-translational modification that modulates biological processes such as the immune response and protein trafficking. Altered glycosylation profiles are associated with cancer and inflammatory diseases, as well as impacting the efficacy of therapeutic monoclonal antibodies. Consisting of oligosaccharides attached to asparagine residues, enzymatically released N-linked glycans are analytically challenging due to the diversity of isomeric structures that exist. A commonly used technique for quantitative N-glycan analysis is liquid chromatography-mass spectrometry (LC-MS), which performs glycan separation and characterization. Although many reversed and normal stationary phases have been utilized for the separation of N-glycans, porous graphitic carbon (PGC) chromatography has become desirable because of its higher resolving capability, but is difficult to implement in a robust and reproducible manner. Herein, we demonstrate the analytical properties of a 15 cm fused silica capillary (75 µm i.d., 360 µm o.d.) packed in-house with Hypercarb PGC (3 µm) coupled to an Agilent 6550 Q-TOF mass spectrometer for N-glycan analysis in positive ion mode. In repeatability and intermediate precision measurements conducted on released N-glycans from a glycoprotein standard mixture, the majority of N-glycans reported low coefficients of variation with respect to retention times (≤4.2%) and peak areas (≤14.4%). N-glycans released from complex samples were also examined by PGC LC-MS. A total of 120 N-glycan structural and compositional isomers were obtained from formalin-fixed paraffin-embedded ovarian cancer tissue sections. Finally, a comparison between early- and late-stage formalin-fixed paraffin-embedded ovarian cancer tissues revealed qualitative changes in the α2,3- and α2,6-sialic acid linkage of a fucosylated bi-antennary complex N-glycan. Although the α2,3-linkage was predominant in late-stage ovarian cancer, the alternate α2,6-linkage was more prevalent in early-stage ovarian cancer. This study establishes the utility of in-house packed PGC columns for the robust and reproducible LC-MS analysis of N-glycans.

Quantitative Approach Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-ToF) Mass Spectrometry.

Quantitation using mass spectrometry (MS) is a routine approach for multiple analytes, including small molecules and peptides. Electrospray-based MS platforms are typically employed, as they provide highly reproducible outputs for batch processing of multiple samples. Quantitation using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF) mass spectrometry, while less commonly adopted, offers the ability to monitor analytes at significantly higher throughput and lower cost compared with ESI MS. Achieving accurate quantitation using this approach requires the development of appropriate sample preparation, spiking of appropriate internal standards, and acquisition to minimize spot-to-spot variability. Here we describe the preparation of samples for accurate quantitation using MALDI-ToF MS. The methodology presented shows the ability to quantitate perfluorooctanesulfonic acid (PFOS) from contaminated water.

Scavenging of soluble and immobilized CCL21 by ACKR4 regulates peripheral dendritic cell emigration.

Leukocyte homing driven by the chemokine CCL21 is pivotal for adaptive immunity because it controls dendritic cell (DC) and T cell migration through CCR7. ACKR4 scavenges CCL21 and has been shown to play an essential role in DC trafficking at the steady state and during immune responses to tumors and cutaneous inflammation. However, the mechanism by which ACKR4 regulates peripheral DC migration is unknown, and the extent to which it regulates CCL21 in steady-state skin and lymph nodes (LNs) is contested. Specifically, our previous findings that CCL21 levels are increased in LNs of ACKR4-deficient mice [I. Comerford et al., Blood 116, 4130-4140 (2010)] were refuted [M. H. Ulvmar et al., Nat. Immunol. 15, 623-630 (2014)], and no differences in CCL21 levels in steady-state skin of ACKR4-deficient mice were reported despite compromised CCR7-dependent DC egress in these animals [S. A. Bryce et al., J. Immunol. 196, 3341-3353 (2016)]. Here, we resolve these issues and reveal that two forms of CCL21, full-length immobilized and cleaved soluble CCL21, exist in steady-state barrier tissues, and both are regulated by ACKR4. Without ACKR4, extracellular CCL21 gradients in barrier sites are saturated and nonfunctional, DCs cannot home directly to lymphatic vessels, and excess soluble CCL21 from peripheral tissues pollutes downstream LNs. The results identify the mechanism by which ACKR4 controls DC migration in barrier tissues and reveal a complex mode of CCL21 regulation in vivo, which enhances understanding of functional chemokine gradient formation.

Quantitative mass spectrometry-based analysis of proteins related to cattle and their products - Focus on cows' milk beta-casein proteoforms.

Modern mass spectrometers can accurately measure thousands of compounds in complex mixtures over a given liquid chromatograph method, depending on desired outcome and method duration. This stream of analytical chemistry has wide ranging application across food, pharma, environmental, forensics, clinical and research. With consistent pressure on both the ruminant production and product industries to face new and substantial challenges, liquid chromatography-mass spectrometry (LC-MS) is an ideal tool to identify, detect and quantify markers of breeding, production and adaption to support both research and industry to overcome these challenges. Herein, we provide a description of the theoretical basis and framework for LC-MS as a rapidly developing technique and highlight its application in measuring cattle and cattle product traits through protein quantitation with specific focus on beta-casein proteoforms.

Gelatin-coated indium tin oxide slides improve human cartilage-bone tissue adherence and N-glycan signal intensity for mass spectrometry imaging.

Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) has been successfully used to elucidate the relative abundance and spatial mapping of analytes in situ. Currently, sample preparation workflows for soft formalin-fixed paraffin-embedded (FFPE) tissues, such as brain, liver, kidney, and heart, have been successfully developed. However, hard tissues, such as cartilage-bone, tooth, and whole mouse body, have resulted in the loss of morphology or tissue during the heat-induced epitope retrieval (HIER) step on commercially available conductive indium tin oxide (ITO) slides. Therefore, we have successfully developed a novel and cost-effective sample preparation workflow in which commercial conductive ITO slides are pre-coated with gelatin and chromium potassium sulfate dodecahydrate to improve the adherence of FFPE human osteoarthritic cartilage-bone tissue sections. Gelatin-coated ITO slides also resulted in overall higher N-glycan signal intensity for not only FFPE osteoarthritic cartilage-bone tissue but also for FFPE hard-boiled egg white used as a quality control to assess the quality of sample preparation and MALDI-MSI acquisition. In summary, we present a novel straightforward workflow to improve slide adherence and morphological preservation of FFPE cartilage-bone tissue sections during HIER while improving the signal intensity of N-glycans spatially mapped from the same tissue sections by MALDI-MSI.

Novel technical developments in mass spectrometry imaging in 2020: A mini review.

The applicability of mass spectrometry imaging (MSI) has exponentially increased with the improvement of sample preparation, instrumentation (spatial resolution) and data analysis. The number of MSI publications listed in PubMed continues to grow with 378 published articles in 2020-2021. Initially, MSI was just sensitive enough to identify molecular features correlating with distinct tissue regions, similar to the resolution achieved by visual inspection after standard immunohistochemical staining. Although the spatial resolution was limited compared with other imaging modalities, the molecular intensity mapping added a new exciting capability. Over the past decade, significant improvements in every step of the workflow and most importantly in instrumentation were made, which now enables the molecular analysis at a cellular and even subcellular level. Here, we summarize the latest developments in MSI, with a focus on the latest approaches for tissue-based imaging described in 2020.

Mass Spectrometry Imaging as a Potential Tool to Investigate Human Osteoarthritis at the Tissue Level.

Osteoarthritis (OA) is the most common degenerative joint disease, predicted to increase in incidence year by year due to an ageing population. Due to the biological complexity of the disease, OA remains highly heterogeneous. Although much work has been undertaken in the past few years, underlying molecular mechanisms leading to joint tissue structural deterioration are not fully understood, with only few validated markers for disease diagnosis and progression being available. Discovery and quantitation of various OA-specific biomarkers is still largely focused on the bodily fluids which does not appear to be reliable and sensitive enough. However, with the advancement of spatial proteomic techniques, several novel peptides and proteins, as well as N-glycans, can be identified and localised in a reliable and sensitive manner. To summarise the important findings from OA biomarker studies, papers published between 2000 and 2020 were searched via Google Scholar and PubMed. Medical subject heading (MeSH) terms 'osteoarthritis', 'biomarker', 'synovial fluid', 'serum', 'urine', 'matrix-assisted laser desorption/ionisation', 'mass spectrometry imaging', 'proteomic', 'glycomic', 'cartilage', 'synovium' AND 'subchondral bone' were selectively used. The literature search was restricted to full-text original research articles and written only in English. Two main areas were reviewed for OA biomarker studies: (1) an overview of disease-specific markers detected from different types of OA bio-samples, and (2) an up-to-date summary of the tissue-specific OA studies that have utilised matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI). Overall, these OA biomarkers could provide clinicians with information for better the diagnosis, and prognosis of individual patients, and ultimately help facilitate the development of disease-modifying treatments.

Egg White as a Quality Control in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI).

The strength of MALDI-MSI is to analyze and visualize spatial intensities of molecular features from an intact tissue. The distribution of the intensities can then be visualized within a single tissue section or compared in between sections, acquired consecutively. This method can be reliably used to reveal physiological structures and has the potential to identify molecular details, which correlate with biological outcomes. MALDI-MSI implementation in clinical laboratories requires the ability to ensure method quality and validation to meet diagnostic expectations. To be able to get consistent qualitative and quantitative results, standardized sample preparation and data acquisition are of highest priority. We have previously shown that the deposition of internal standards onto the tissue section during sample preparation can be used to improve the mass accuracy of monitored m/z features across the sample. Here, we present the use of external and internal controls for the quality check of sample preparation and data acquisition, which is particularly relevant when either many spectra are acquired during a single MALDI-MSI experiment or data from independent experiments are processed together. To monitor detector performance and sample preparation, we use egg white as an external control for peptide and N-glycan MALDI-MSI throughout the experiment. We have also identified endogenous peptides from cytoskeletal proteins, which can be reliably monitored in gynecological tissue samples. Lastly, we summarize our standard quality control workflow designed to produce reliable and comparable MALDI-MSI data from single sections and tissue microarrays (TMAs).

An external quality assurance trial to assess mass spectrometry protein testing facilities for identifying multiple human peptides.

The application of proteomic liquid chromatography mass spectrometry (LC-MS) for identifying proteins and peptides associated with human disease is rapidly growing in clinical diagnostics. However, the ability to accurately and consistently detect disease-associated peptides remains clinically uncertain. Variability in diagnostic testing occurs in part due to the absence of appropriate reference testing materials and standardised clinical guidelines for proteomic testing. In addition, multiple proteomic testing pipelines have not been fully assessed through external quality assurance (EQA). This trial was therefore devised to evaluate the performance of a small number of mass spectrometry (MS) testing facilities to (i) evaluate the EQA material for potential usage in a proteomic quality assurance program, and to (ii) identify key problem areas associated with human peptide testing. Five laboratories were sent six peptide reference testing samples formulated to contain a total of 35 peptides in differing ratios of light (natural) to heavy (labelled) peptides. Proficiency assessment of laboratory data used a modified approach to similarity and dissimilarity testing that was based on Bray-Curtis and Sorensen indices. Proficiency EQA concordant consensus values could not be derived from the assessed data since none of the laboratories correctly identified all reference testing peptides in all samples. However, the produced data may be reflective of specific inter-laboratory differences for detecting multiple peptides since no two testing pipelines used were the same for any laboratory. In addition, laboratory feedback indicated that peptide filtering of the reference material was a common key problem area prior to analysis. These data highlight the importance of an EQA programme for identifying underlying testing issues so that improvements can be made and confidence for clinical diagnostic analysis can be attained.

MALDI Mass Spectrometry Imaging of Early- and Late-Stage Serous Ovarian Cancer Tissue Reveals Stage-Specific N-Glycans.

Epithelial ovarian cancer is one of the most fatal gynecological malignancies in adult women. As studies on protein N-glycosylation have extensively reported aberrant patterns in the ovarian cancer tumor microenvironment, obtaining spatial information will uncover tumor-specific N-glycan alterations in ovarian cancer development and progression. matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is employed to investigate N-glycan distribution on formalin-fixed paraffin-embedded ovarian cancer tissue sections from early- and late-stage patients. Tumor-specific N-glycans are identified and structurally characterized by porous graphitized carbon-liquid chromatography-electrospray ionization-tandem mass spectrometry (PGC-LC-ESI-MS/MS), and then assigned to high-resolution images obtained from MALDI-MSI. Spatial distribution of 14 N-glycans is obtained by MALDI-MSI and 42 N-glycans (including structural and compositional isomers) identified and structurally characterized by LC-MS. The spatial distribution of oligomannose, complex neutral, bisecting, and sialylated N-glycan families are localized to the tumor regions of late-stage ovarian cancer patients relative to early-stage patients. Potential N-glycan diagnostic markers that emerge include the oligomannose structure, (Hex)6 + (Man)3 (GlcNAc)2 , and the complex neutral structure, (Hex)2 (HexNAc)2 (Deoxyhexose)1 + (Man)3 (GlcNAc)2 . The distribution of these markers is evaluated using a tissue microarray of early- and late-stage patients.

Rapid separation and identification of beer spoilage bacteria by inertial microfluidics and MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS), in combination with Biotyper software, is a rapid, high-throughput, and accurate method for the identification of microbes. Microbial outbreaks in a brewery present a major risk for companies as it can lead to cost-intensive recalls and damage to the brand reputation. MALDI-TOF MS has been implemented into a brewery setting for quality control practices and the identification of beer spoilage microorganisms. However, the applicability of this approach is hindered by compatibility issues associated with mixed cultures, requiring the use of time-consuming selective cultivation techniques prior to identification. We propose a novel, low-cost approach based on the combination of inertial microfluidics and secondary flows in a spiral microchannel for high-throughput and efficient separation of yeasts (Saccharomyces pastorianus and Saccharomyces cerevisiae) from beer spoilage microorganisms (Lactobacillus brevis and Pediococcus damnosus). Flow rates were optimised using S. pastorianus and L. brevis, leading to separation of more than 90% of the L. brevis cells from yeast. The microorganisms were then identified to the species level using the MALDI-TOF MS platform using standard sample preparation protocols. This study shows the high-throughput and rapid separation of spoilage microorganisms (0.3-3 µm) from background yeast (5 µm) from beer, subsequent identification using MALDI Biotyper, and the potential applicability of the approach for biological control in the brewing industry.

Translating N-Glycan Analytical Applications into Clinical Strategies for Ovarian Cancer.

Protein glycosylation, particularly N-linked glycosylation, is a complex posttranslational modification (PTM), which plays an important role in protein folding and conformation, regulating protein stability and activity, cell-cell interaction, and cell signaling pathways. This review focuses on analytical techniques, primarily MS-based techniques, to qualitatively and quantitatively assess N-glycosylation while successfully characterizing compositional, structural, and linkage features with high specificity and sensitivity. The analytical techniques explored in this review include LC-ESI-MS/MS and MALDI time-of-flight MS (MALDI-TOF-MS), which have been used to analyze clinical samples, such as serum, plasma, ascites, and tissue. Targeting the aberrant N-glycosylation patterns observed in MALDI-MS imaging (MSI) offers a platform to visualize N-glycans in tissue-specific regions. The studies on the intra-patient (i.e., a comparison of tissue-specific regions from the same patient) and inter-patient (i.e., a comparison of tissue-specific regions between different patients) variation of early- and late-stage ovarian cancer (OC) patients identify specific N-glycan differences that improve understanding of the tumor microenvironment and potentially improve therapeutic strategies for the clinic.

Raw N-glycan mass spectrometry imaging data on formalin-fixed mouse kidney.

Provided is the annotated raw data for N-glycan mass spectrometry imaging (MSI) annotations in thin cross-sections of formalin-fixed and paraffin-embedded murine kidney. Relevant meta-data have been provided in this brief and the raw MSI data can be accessed using ProteomeXchange with the PRoteomics IDEntifications (PRIDE) identifier PXD009808. This brief is the first in a set of submissions from our group which will make raw data publicly accessible for existing and future MSI studies.

Balancing sufficiency and impact in reporting standards for mass spectrometry imaging experiments.

Reproducibility, or a lack thereof, is an increasingly important topic across many research fields. A key aspect of reproducibility is accurate reporting of both experiments and the resulting data. Herein, we propose a reporting guideline for mass spectrometry imaging (MSI). Previous standards have laid out guidelines sufficient to guarantee a certain quality of reporting; however, they set a high bar and as a consequence can be exhaustive and broad, thus limiting uptake.To help address this lack of uptake, we propose a reporting supplement-Minimum Information About a Mass Spectrometry Imaging Experiment (MIAMSIE)-and its abbreviated reporting standard version, MSIcheck. MIAMSIE is intended to improve author-driven reporting. It is intentionally not exhaustive, but is rather designed for extensibility and could therefore eventually become analogous to existing standards that aim to guarantee reporting quality. Conversely, its abbreviated form MSIcheck is intended as a diagnostic tool focused on key aspects in MSI reporting.We discuss how existing standards influenced MIAMSIE/MSIcheck and how these new approaches could positively impact reporting quality, followed by test implementation of both standards to demonstrate their use. For MIAMSIE, we report on author reviews of four articles and a dataset. For MSIcheck, we show a snapshot review of a one-month subset of the MSI literature that indicated issues with data provision and the reporting of both data analysis steps and calibration settings for MS systems. Although our contribution is MSI specific, we believe the underlying approach could be considered as a general strategy for improving scientific reporting.

Quantitation and Identification of Intact Major Milk Proteins for High-Throughput LC-ESI-Q-TOF MS Analyses.

Cow's milk is an important source of proteins in human nutrition. On average, cow's milk contains 3.5% protein. The most abundant proteins in bovine milk are caseins and some of the whey proteins, namely beta-lactoglobulin, alpha-lactalbumin, and serum albumin. A number of allelic variants and post-translationally modified forms of these proteins have been identified. Their occurrence varies with breed, individuality, stage of lactation, and health and nutritional status of the animal. It is therefore essential to have reliable methods of detection and quantitation of these proteins. Traditionally, major milk proteins are quantified using liquid chromatography (LC) and ultra violet detection method. However, as these protein variants co-elute to some degree, another dimension of separation is beneficial to accurately measure their amounts. Mass spectrometry (MS) offers such a tool. In this study, we tested several RP-HPLC and MS parameters to optimise the analysis of intact bovine proteins from milk. From our tests, we developed an optimum method that includes a 20-28-40% phase B gradient with 0.02% TFA in both mobile phases, at 0.2 mL/min flow rate, using 75°C for the C8 column temperature, scanning every 3 sec over a 600-3000 m/z window. The optimisations were performed using external standards commercially purchased for which ionisation efficiency, linearity of calibration, LOD, LOQ, sensitivity, selectivity, precision, reproducibility, and mass accuracy were demonstrated. From the MS analysis, we can use extracted ion chromatograms (EICs) of specific ion series of known proteins and integrate peaks at defined retention time (RT) window for quantitation purposes. This optimum quantitative method was successfully applied to two bulk milk samples from different breeds, Holstein-Friesian and Jersey, to assess differences in protein variant levels.