Poster Abstracts

P1) diaPASEF: Toward the Ideal Mass Analyzer with Data-Independent Acquisition and Parallel Accumulation-Serial Fragmentation

Presenting Author: Frederic Lamoliatte (Bruker Daltonics, Coventry, UK)

Other Authors: Florian Meier, Andreas-David Brunner, Annie Ha, Max Frank, Eugenia Voytik, Stephanie Kaspar-Schoenefeld, Markus Lubeck, Heiner Koch, Scarlet Koch, Oliver Raether, Frederic Lamoliatte, Ruedi Aebersold, Ben Collins, Hannes Rost, Matthias Mann

Introduction 

DIA promises reproducible and accurate protein quantification across large sample cohorts. Current methods utilise only about 1-3% of all available ions. In principle, all ions could be utilised by parallel ion storage and sequential release from the TIMS device into a Q-TOF mass analyser. Here, we asked if the PASEF principle could be transferred to DIA.

Methods 

We tested multiple schemes for precursor selection window size and placement in the m/z-ion mobility plane. Analysis of the four-dimensional data space has been incorporated into OpenSWATH. For ion mobility-aware targeted data extraction, we used a project-specific library from 48 high-pH reverse-phase peptide fractions acquired with PASEF.

Results

We derived multiple diaPASEF acquisition schemes from the density distribution of about 130,000 precursors present in the library. After linear alignment, CCS values extracted from the diaPASEF runs deviated < 2% from the library. In triplicate 120min runs of 200ng HeLa digest each, we quantified over 7000 proteins at a 1% FDR. Fragment ion-based quantification was very reproducible with a median CVs of 10% and a pairwise mean Pearson correlation >0.96. 

Conclusion

The diaPASEF method captures and utilises a very large proportion of the available ion current, approaching the ideal mass analyser.



P2) Regulation of Cell Metabolism by LAR, a Receptor-Type Protein Tyrosine Phosphatase

Presenting Author: Carys Howell (University of Birmingham)

Other Authors: Ruth Walker, Neil Hotchin and Debbie Cunningham

Phosphorylation is a key post-translational protein modification that is regulated by kinases and phosphatases which, respectively, add or remove phosphate groups from the amino acids serine, threonine and tyrosine of their substrates. There is a wealth of literature surrounding the biological processes regulated by kinases, yet we know very little about the events regulated by phosphatases. We are interested in the protein tyrosine phosphatase, leukocyte antigen related protein (LAR), also known as protein tyrosine phosphatase receptor type F (PTPRF). LAR is widely expressed and is known to regulate neuronal development, cell adhesion, mammary gland formation and insulin signalling. LAR consists of an extracellular domain, a transmembrane domain and an intracellular domain that has phosphatase activity. Our recent quantitative proteomics study compared WT mouse embryonic fibroblasts (MEFs) to MEFs with the phosphatase domain removed (LARP MEFs). This study highlighted that many proteins with mitochondrial/metabolic function were significantly up/down regulated in LARP MEFs. Here, we present data indicating that LARP MEFs show significantly reduced respiratory activity in comparison to WT MEFs. These results highlight a role for LAR as a regulator of mitochondrial activity and will assist in unravelling the role of LAR as novel a regulator of cellular metabolism.



P3) Rapid Screening of Proteins Adsorbed on Biomaterial Surfaces Using LESA-MS/MS 

Presenting Author: Joris Meurs (University of Nottingham)

Other Authors: Morgan R. Alexander, David A. Barrett & Dong-Hyun Kim

Pluripotent stem cells are a valuable source for cell production and have a huge potential to serve for a multitude of applications in regenerative medicine. Biomaterials have been discovered that assist as synthetic substrates for pluripotency maintenance during expansion, but the mechanism by which they achieve this is not well understood because of poor characterisation of the biointerface. Pre-adsorption of proteins from the culture medium is believed to be a crucial element in this process. Current analytical methodologies are limited in throughput and therefore not compatible with achieving a broad assessment of a number of materials in high throughput biomaterials discovery. Here, we used liquid extraction surface analysis-tandem mass spectrometry (LESA-MS/MS) as a high throughput tool for the analysis of surface-adsorbed proteins. In combination with microwave-assisted in situ trypsin digestion, this analysis strategy reduces the total workflow time ~100-fold compared to a standard LC-MS/MS protocol. Furthermore, similar digestion efficiencies were found for a set of protein standards when comparing microwave-assisted in situ digestion with in-solution digestion. The next step is to employ the methodology on biomaterial microarrays incubated in medium.




P4) Method Development for the Identification of Proteins in Fingertip Smears using MALDI-MS 

Presenting Author: Cristina Russo (Sheffield Hallam University)

Other Authors: Laura Cole, Lynda Wyld, Simona Francese

Matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS) is a soft ionisation mass spectrometry technique widely employed for the detection of large biomolecules (proteins and peptides).

Previous studies have described the use of MALDI-MS for fingermark analysis of proteins that the literature has indicated as potential biomarkers for breast cancer. Fingertip smears can represent a significant opportunity to devise a non-invasive screening of biomarkers for prognostic and diagnostic purposes.

Prior to investigation of strong candidates for breast cancer biomarkers in fingertip smears by using MALDI-MS tools, proteolytic condition optimisation is required in order to maximise the potential to recover cancer biomarkers.

In this study different approaches have been investigated to detect strong candidates for breast cancer biomarkers in fingertip smears by using MALDI-MS based methods




P5) Forensic Detection of Haemoglobin Variants through MALDI Mass Spectrometry of Blood

Presenting Author: Cameron Heaton (Sheffield Hallam University)

Other Authors: Laura Cole, Richard McColm, Jason Eyre, Simona Francese

Blood is commonly encountered at the scene of violent crime. Conventional techniques for blood analysis are typically destructive, such as swabbing and extracting analytes. Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI MS) is only partially-destructive and can have subsequent enhancement techniques applied post-analysis. This is desirable for example in maintaining ridge detail in bloody fingerprints. There are >1300 haemoglobin variants in a small percentage of the human population, cause by amino acid mutations in the haemoglobin sub-unit chains, such as sickle cell disease. This research will explore discriminating blood of different haemoglobin variant carriers, using MS to detect the different peptides as a result of these mutations. It is hoped that this could be used to help identify or exonerate suspects or victims that have had blood recovered from a crime scene.




P6) Quadrupole Theory Explored using LabVIEW 

Presenting Author: Tania Karasiewicz (Nottingham Trent University)

Other Authors: David Kilgour

In this poster, I will show how you can use simple methods to look at ion secular frequencies in quadrupoles and ion traps, by two different methods: modelling the ion motion and solving the beta equation. I will then use these methods to compare the Mathieu stability diagrams generated by both techniques.



P7) Investigation of Induction of Xenobiotic Metabolising Enzymes in a 3D Skin Model by using RT-qPCR and GeLC-MS/MS Techniques.

Presenting Author: Hatem Sallem (Sheffield Hallam University)

Other Authors: Tanya Klymenko, Catherine Duckett, Vikki A Carolan and Malcolm R Clench

Skin is the largest organ in the body and is used as a defence against xenobiotic (foreign chemical compounds). Xenobiotic metabolism enzymes (XME) play a significant role in an organism's defence against potentially toxic chemicals. It is important to measure the levels of phase I and II drug metabolism enzymes in 3D skin models that used in safety evaluation studies such as, pharmaceuticals, cosmetic and toxicity. Overall, the drug metabolism takes place in two phase reactions: Phase I drug metabolism reaction (e.g, oxidation, reduction, and hydrolysis) included formation of new or modified function group (oxidation, reduction and hydrolysis) such as OH or COOH. Phase II of drug metabolism (Phase II reactions) includes conjugation with an endogenous substance (e.g, glucuronic acid, sulfate, and glycine); these reaction conjugate with phase I which make drug are more polar (water soluble) and thus more readily excreted from the body.

Human and 3D skin models has been treated with a series of inducers of the metabolising enzymes such as, β-Naphthoflavone (β-NF), phenobarbital, all -trans- retinoic acid to metabolising enzyme tested. The workflow involves electrophoresis on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). After SDS-PAGE, the gel will be stained for visualising protein lanes and each band slices cut into 1mm3 fractions along the length of the gel, then each slide fraction will be digested with trypsin to create peptides. Following purification these will be analysed by direct infusion electrospray mass spectrometry. 

The purpose of this study is to compare levels of the major phase I (including cytochrome P45-dependentmonooxgenase, flavin-containing monooxygenase and esterases), and phase II metabolizing enzymes (including glutathione-S-transferase and N-Acetyltransferase) expressed in a commercially available 3D skin model, ""Labskin"", and human skin using by RT-qPCR and GeLC-MS/MS techniques. In this present study will be collected the data information regarding Progenesis QI for proteomics mass spectrum that involved the most important XMEs.



P8) Biomarkers for Heart Failure in BIOSTAT cohort: A PRM approach

Presenting Author: Shimon Atunde (University of Leicester)

Heart Failure (HF) is a clinical syndrome caused by structural and functional abnormalities in the myocardium. These defects lead to difficulties of ventricular fill and blood ejection (Inamdar and Inamdar, 2016). An ischemic event that principally affects the left ventricle is often the cause behind HF leading to symptoms that are often overlapping with other medical conditions especially in the elderly; the part of the population most susceptible to heart failure (Krum and Driscoll, 2013). Symptoms of HF include dyspnea, orthopnea and paroxysmal nocturnal dyspnea (Inamdar and Inamdar, 2016). 

HF is a serious public health issue affecting over 23 million individuals worldwide (Roger, 2013). Cowie (2017) describes HF as an epidemic affecting over 500,000 individuals and accounting for 1-2% of health expenditure in the United Kingdom (UK) alone. These findings emphasise the importance of HF and why research into a better understanding of the syndrome is essential.

Research by Cao et al in the Department of Cardiovascular Sciences at the University of Leicester identified over 2000 proteins in the plasma samples of 85 “alive” and “dead” heart failure (HF) patients from the BIOSTAT cohort using ESI-MS. 

Methods: To choose which of the identified proteins to target in plasma samples, different statistical techniques will be employed including sparse principal component analysis and sparse partial least squares discrimination analysis (combined with Cytoscape analysis). These statistically selected proteins will be measured in the same 85 patients from within the BIOSTAT cohort using a targeted proteomics approach. Plasma of the chosen patients will undergo digestion and the tryptic peptides detected and measured using liquid chromatography combined with mass spectrometry using parallel reaction monitoring (LC-MS-PRM). LC-MS-PRM will be combined with labelled standards for accurate quantification of these proteins. 

Discussion: This project will contribute to a greater understanding of HF pathophysiology particularly in terms of potential biomarkers in HF. Biomarkers in HF have the potential to detect this disease early and therefore, provide an opportunity for early intervention and personalized care for patients. This validated set of proteins will confirm the suitability of these proteins as biomarkers for the prognosis of HF and possibly allow a better understanding of the underlying pathophysiology and aetiology of heart failure.



P9) Regulation of Synaptic Protein Function by the Lysine Deacetylase Sirtuin-2

Presenting Author: Hatoon Alamri (University of Sheffield)

Other Authors: Mark Collins

Recent studies indicate an emerging role of lysine acetylation in the regulation of protein stability. Acetylation is a reversible process controlled by families of (KAT) and (KDAC) enzymes. It is particularly enriched in the brain compared to other tissues and recent studies highlight and important regulatory role of acetylation in several neurophysiological processes. 

SIRT2 is a NAD-dependent KDAC enzyme associated with several neurodegenerative diseases and is highly expressed in the brain. Sirt2 deacetylation of the AMPA receptor, Arc and CDK5 regulate their stability, turnover, and their function in synaptic plasticity. The mechanism of this regulation lies in the fact that Sirt2 maintains the balance between acetylation and ubiquitination at individual lysine residues; deacetylation by Sirt2 reveals lysine residues that are then ubiquitinated leading to degradation of the protein.

In order to identify novel substrates of Sirt2, we have enriched acetylated peptides from tryptic digests of WT and Sirt2 KO mouse brain tissue and analysed them using quantitative mass spectrometry. Using this strategy, we identified more than 1500 proteins acetylated on more than 2000 sites in mice brain tissue, of which 250 sites were significantly upregulated in the absence of Sirt2. Selected putative substrates are now being validated using orthogonal approaches. 



P10) RaPID Digestion Method for Bottom-up Proteomics 

Presenting Author: Dhanwin Baker (Nottingham Trent University)

Other Authors: Clare Coveney, Jon Oyler, David Boocock, David Kilgour

Bottom-up proteomics methods are commonly used for a very wide variety of analysis in biomedical and pharmaceutical research. There are three frequently reported weaknesses in the most commonly used bottom-up digestions methods: the time taken by the digestion step is long (often of the order of 18-24 hours); the methods can be difficult to automate; and there can be issues with repeatability. We are developing a bottom-up proteomic digestion method (RaPID) that is showing good results even when the protein digestion step is reduced from traditional 18-24 hours to as little as 5 minutes. This process involves a polar organic solvent, such as acetonitrile, added at an optimum concentration, followed by the digestion step carried out at an optimized temperature. Fibrinogen and Human protein Promega K562 have been used as test proteins for these experiments. The LC-MS datasets produced have been processed using both commercial and open source proteomic software with varying algorithms. Current results indicate the importance of robust temperature control and suggest that reduction and alkylation steps may be required for significant sequence coverage on some proteins. Further experiments need to be performed to establish whether this new method could be a universal method for protein digestion.



P11) Progress in Biopharmaceutical Detection in an Aggregated 3D Cell Culture Model by Mass Spectrometry Imaging

Presenting Author: Lucy Flint (Sheffield Hallam University)

Other Authors: Gregory Hamm, Neil A. Cross, Laura M. Cole, David P. Smith, Richard J. A. Goodwin and Malcolm R. Clench

A growing priority in the pharma-industry is to effectively analyse the delivery of complex biopharmaceuticals in situ. Here, a mass spectrometry imaging (MSI) platform has been developed to directly map the distribution of a therapeutic antibody, Cetuximab in a 3D lung adenocarcinoma cell culture model. We developed an aggregated spheroid model 'aggregoid' (~1 mm diameter) to represent an in vivo tumour for the study of drug distribution and response. Initial imaging analysis has observed endogenous metabolite and lipid distributions at high spatial resolution to enable understanding of the aggregoid's structure and the biological response to Cetuximab treatment.

To identify the distribution of Cetuximab in the 3D lung adenocarcinoma aggregoid model, a bottom-up proteomics approach was designed to identify the monoclonal antibody by its unique peptides. An experimental design was employed to optimise parameters including trypsin solution, trypsin deposition, sample washing, and matrix application for a robust on-tissue digest technique to achieve high quality peptide images. The developed method assisted the detection of Cetuximab peptides and endogenous protein-originated peptides within dosed aggregoids at 24 hr and 96 hr. The on-tissue digest methodology was successfully used to observe proteotypic imaging peptides derived from Cetuximab and endogenous proteins by both MALDI- and DESI-MSI.




P12) Accelerating SWATH® Acquisition for Protein Quantitation – Up to 100 Samples per Day

Presenting Author: Rebekah Sayers (Sciex UK)

Other Authors: Christie Hunter and Nick Morrice

The combination of microflow LC with SWATH Acquisition for accelerating quantitative proteomics studies is becoming increasingly more widespread, due to the improved robustness and throughput obtained relative to the traditional nanoflow LC approach. Previously an investigation was done to characterize the tradeoff between sensitivity and throughput/robustness when switching from nanoflow to microflow for global protein quantitation experiment using SWATH Acquisition. It was demonstrated that with only 4x more total protein on column similar results could be achieved with much higher throughput and robustness. The impact of shortening microflow gradient length on the number of proteins/peptides quantified from a SWATH acquisition dataset was also studied, providing a good guidance for researchers when selecting the optimal chromatographic strategy for a study. Due to these method optimizations, microflow SWATH acquisition datasets can be generated with high throughput (up to 100 samples per day, depending on the selected gradient length). This will put high pressure on the downstream data processing tools and results generation, to keep pace with the generation of datasets. Thus, processing of Omics datasets in the cloud is becoming increasingly important, to handle the speed and scale of today’s industrialized proteomics approaches.



P13) Optimisation of Trypsin Concentration for MALDI-MS Based Analysis of Blood Marks.

Presenting Author: Katie Kennedy (Sheffield Hallam University)

Other Authors: Laura Cole, Mark Sealey and Simona Francese

Rapid and confirmatory blood detection through implementation of a MALDI-MS based strategy is proving to be a far more specific test to the conventionally deployed presumptive tests for blood detection at crime scenes. With several variables to account for in blood mark recovery such as deposition surface, utilisation of blood enhancement techniques to aid visualisation of latent blood stains, the age and volume of blood, there is a pressing need for refinement of this MALDI based approach. Investigation into the efficiency of the selected trypsin concentration used with the varying volumes of blood is necessary. In solution digestions of increasing blood volumes using a known concentration of Trypsin have been carried out to ascertain the lower and higher volume thresholds for feasible detection of blood protein signatures at this concentration. These experiments provide guidance as to the appropriate sample preparation protocol for bloodstains and has offered an insight into the robustness of the protein concentration for different quantities of blood that may be found at crime scenes. Development of user-friendly flowcharts for end users is underway to advise the most appropriate sample preparation method for the given blood volume when discovered as both bloodstains and blood finger marks.



P14) LESA Sampling of Human Non-Alcoholic Fatty Liver Disease Tissue for the Profiling of Liver Fatty Acid Binding Protein

Presenting Author: James Hughes (University of Birmingham)

Other Authors: Patricia F. Lalor, Iain B. Styles, Helen J. Cooper.

We demonstrate the use of liquid extraction surface analysis for the rapid profiling of liver fatty acid binding protein and the clinically relevant T94A variant from human liver samples of nonalcoholic fatty liver disease. This chronic disease of the liver can progress to non-alcoholic steatohepatitis and further to cirrhosis and hepatocellular carcinoma. Rapid identification of this variant could be used as a prognostic marker for this disease. Thin tissue sections (10 μm) of human liver from patients (n=32) with a range of NAFLD pathologies were thaw mounted onto glass slides. LESA MS was performed by use of the Advion Triversa Nanomate coupled to the UltraFAIMS Owstone device which in turn was coupled to a Thermo Fisher Orbitrap Elite. Analysis was performed on both washed and unwashed tissue samples. Through use of the UltraFAIMS device and the Nanomate LESA platform, 

FABP1 was extracted and identified from fresh frozen human liver tissue sections. CID, ETD, and HCD were used to fragment the intact protein and confirm the identity and the presence of the variant or wild type. The T94A variant was observed in higher frequency among patients with NAFLD which suggests that the variant could be used as a prognostic marker of disease and LESA MS can provide a rapid and robust method of detection from biopsy tissue samples.



P15) Proteotypic Peptides for MS Imaging Applications

Presenting Author: Malcolm Clench (Sheffield Hallam University)

Other Authors: Laura M Cole, David P Smith, Neil Cross, Emily Humphrys, Laurent Caron.

The concept of proteotypic peptides is well understood in conventional proteomics and such peptides are routinely synthesised in unlabelled and stable isotope labelled formats for use in MRM type quantitative proteomics experiments. In 2013 we published an article describing the production of recombinant "IMS TAG" proteins as a new method for validating bottom‐up matrix‐assisted laser desorption/ionisation ion mobility separation mass spectrometry imaging [1]. This paper described synthesis of a recombinant protein (as a variation of the QConCat technique [2]) that when cleaved with trypsin yields a range of peptide standards for use as identification and quantification markers for multiple proteins in one MALDI‐IMS‐MSI experiment. Mass spectrometry images of the distribution of proteins in fresh frozen and formalin‐fixed paraffin‐embedded tissue samples following in situ tryptic digestion were generated by isolating signals on the basis of their m/z value and ion mobility drift time, which were correlated to matching peptides in the recombinant standard. 

In our recent work an examination of the literature on peptides produced in "on-tissue" digest experiments combined with MSI has been carried out.  From this survey a panel of 756 proteotpyic peptides observed in MSI imaging experiments was identified.  These have been collated into a freely searchable on-line database available

 

Recent advances in peptide synthesis have however made the use of a QConCat approach for obtaining standard peptides unnecessary.  Working with Cambridge Research Biochemicals (CRB) a catalogue of peptides from the database has been created and added to CRB's discovery peptide range.  This poster describes the characterisation by MALDI-IMS-MSI of a number of these peptides.

[1] Cole L.M et al Rapid Commun Mass Spectrom 2013, 27, 2355-2362

[2] Pratt J.M  et al Nat Protoc. 2006;1(2):1029-43.



P16) Characterising a Highly Developed 3D Model of Cerebral Cortical Neurons Using Mass Spectrometry and Artificial Neural Network Inference

Presenting Author: Nick Weir (Nottingham Trent University)

Other Authors: Wagner, S., Miles, A.K., Ball, G.R., Stevens, R., Hargreaves, A., McGinnity, T.M., Tinsley, C. J.

Whilst two dimensional (2D) culture is typically employed within the pharmaceutical industry, culture of cells in a three dimensional (3D) environment induces generates an increasingly physiological response from cells. 

Primary cortical neurons cultured on bespoke hydrophobic aligned nanofibre membranes generate self-assembling 3D cellular aggregates with a compartmentalised cell body/neurite structure that is typical of neurons in vivo within 10 days. Due to the physiological morphologies of these neuronal aggregates, mass spectrometry and artificial neural network inference (ANNI) was performed to determine if they were more developed than the control and determine a mechanism for the observed changes. Results indicated that the bespoke fabricated membranes induced a more physiological developmental profile than is typically observed for primary cortical neurons in vitro. ANNI indicated that the mechanism that drives these changes to be synapse dependent, induced by the increased number of synapses that can occur due to the close proximity of neurons within the cluster. 

By improving the quality of in vitro models of the cerebral cortex and developing a facile method of generating organoids, this technology should enable a reduction in the number of animals used in research by increasing the reliability of in vitro data prior to in vivo testing.