69th ASMS conference

Join Evosep at ASMS 2021

Meet Evosep at the orange corner

This year we are back at ASMS, the largest annual gathering of mass spectrometry scientists. Once again we can meet face-to-face and we are excited to learn more about the current needs and research being published in the community. 

All week you can meet us at the orange Evosep corner and we would love to meet you and learn more about you and your current research. We are also more than happy to introduce you to our technology. 

Scientific presence

Our booth is not the only place to learn more about Evosep and the many research posibilities available.  We are excited to advocate several interesting customer talks during this year’s ASMS week.

Learn more about them below and find the full list of all abstracts featuring Evosep One.

 

Talks

Pre-formed gradients for high-throughput and robust nanoflow lipidomics with trapped ion mobility and PASEF

Talk by Florian Meier, Junior Professor at Friedrich-Schiller-Universität Jena
Liquid chromatography – mass spectrometry (LC-MS)-based lipidomics is almost exclusively performed with analytical flow rates due to its throughput and robust operation. However, higher flow rates and larger column diameters lower the electrospray efficiency and hence sensitivity. To increase throughput and robustness of nanoflow separation, the Evosep One LC system elutes samples from disposable trap columns and embeds the analytes in a pre-formed gradient, which is then pushed through the separation column with a single high-pressure pump (PMID: 30104208). Here, we adapt this system to the specific challenges of lipidomics and evaluate its performance in combination with trapped ion mobility spectrometry (TIMS) and deep MS/MS analysis in single runs by parallel accumulation – serial fragmentation (PASEF, PMID: 31949144).
When: Thursday Ocotber 4th at the Lipidomics session: New MS Technologies and Applications 

Evosep One Coupled to the Agilent 6495C QQQ: A robust, sensitive and high-throughput solution for protein biomarker quantitation

Talk by Wendi Hale, Ph.D., LC/MS Applications Scientist at Agilent
Part of the Agilent Breakfast seminar Shortcut to results: Cost reduction and productivity gains through lab automation

Register here

When: Wednesday November 3 at 7.00-8.15 am, Agilent Breakfast session, Room 120 AB

Ultrafast bacterial detection in Urine using high-throughput proteomics and artificial intelligence

Talk by Florence Roux, Project manager – Proteomics and mass spectrometry – chez CHU de Québec-Université Laval

According to the World Health Organization, antibiotic resistance is one of the biggest threats to global health, food security, and development today. To limit the emergence and spreading of new resistances, fast diagnostic tools needs to be developed in order to avoid the overprescription of broad-spectrum antibiotics. Low cost and fast MALDI-TOF MS technology has become an approach of choice for bacterial identification. However, it has several drawbacks: it requires a long step of bacterial culture prior to analysis (>24h), it has a low specificity and is not quantitative.

When: Wednesday November 3 at the Artificial Intelligence in the MS Instrumentation and Applications session

A complete and automated sample preparation strategy for high throughput and standardized proteomics

Talk by Laurent Rieux, Product Specialist at Evosep
Mass spectrometry-based proteomics is fast growing and provides a powerful set of technologies, with the potential to revolutionize health care and enable precision medicine. The Evosep One is specifically designed for high throughput applications, with a focus on clinical analysis of large sample cohorts.For a widespread adoption of proteomics in the clinic, the entire workflow from sample preparation through LC-MS and data analysis needs to be fast and robust. To meet these requirements, we introduce an end-to-end workflow from protein lysate to peptides loaded on Evotips ready for injection on the Evosep One. Furthermore to facilitate high throughput analysis in the clinical laboratory, we have developed a new standardized method the Evosep One allowing 500 samples analyzed per day.
When: Tuesday November 2 at the Artificial Intelligence the High Throughput MS session

Abstracts

This year you can explore a lot of new research featuring Evosep One at ASMS. Below you can find a complete list of all the submitted abstract. Use the search function to filter based on author, organization or topic.

TitleSubjectMaterialYearSummaryAuthorsOrganizationEvosep methodMS instrumentationLearn Moredoc_tag_hfilterevosep_method_hfilterms_vendor_hfilter
TP 021 – Deep and high-throughput urine proteomics profiling for the classification of Parkinson’s disease patientsParkinson’s disease (PD) is the second most abundant neurodegenerative disease (after Alzheimer’s disease) with a prevalence of about 1% for over 60-year-olds. The Parkinson’s Progression Markers Initiative (PPMI) is an observational clinical study for the monitoring of PD progression and identification of new biomarkers. It comprises about 1500 patients in the initial cohort and close to 5000 patients scheduled for an expansion of the study. Previous studies using smaller cohorts have shown that urinary proteome profiling is a promising strategy for the detection of novel biomarkers and patient stratification. We employed our automated high-throughput mass spectrometry (MS)-based proteomics pipeline to the PPMI cohort to identify novel biomarkers for PD.Johannes B Mueller-Reif; Philipp E. Geyer; Sophia Doll; Felix M Riese; Shalini Padmanabhan; Kalpana Merchant; Magnus Schwoerer; Sebastian Virreira WinterOmicEra Diagnostics GmbH; The Michael J. Fox Foundation for Parkinson’s Research; Northwestern University Feinberg School of Medicine
MP 035 – Opportunities and challenges for scaling quantitative proteomics studies with high-throughput DIA-MSQuantitative mass spectrometry is increasingly being used in high-throughput proteomics studies, with ever increasing cohorts and sample sizes. These studies are in part enabled by data independent acquisition (DIA/SWATH) to systematically measure peptides by label-free quantification (LFQ), which gives comprehensive peptide detection and quantification at theoretically unlimited throughput. However, there is limited literature detailing the quantitative challenges of performing proteomics at 100s of samples per week, with most studies focused on the number of detections which are reported. We set out to deeply characterize a high-throughput DIA-MS workflow and assess, empirically, whether accounting for technical variance with quality control and system suitability can be used to improve the accuracy and precision of quantitative proteomics experiments.Lindsay K Pino; Rico Meinl; Erin Broderick; Noelle Schumacker; Daniele Canzani; William E Fondrie; Alexander FederationTalus Bioscience
WP 330 – Beyond TurboTMT: Phi-SDM Super-resolution Methods for Next-generation Highly-multiplexed Quantitative Ultrasensitive and Single-Cell Proteomics via TMTPro Complement Ion DeconvolutionSingle-cell Proteomics has emerged as an extremely promising new field capable of unlocking the vast cellular heterogeneity that defines biology, disease, and therapeutic response. However, it presents enormous challenges, including maximizing both quantitative signal and experimental throughput. We utilize the multiplexed method (e.g., SCOPE-MS/SCOPE2) in which individual cells’ proteomes are labeled, pooled, and combined with abundant carrier material using TMT tags, improving both peptide identification and cell throughput. Still, accurate quantitation via TMT reporter ions remains challenging, in part due to the co-isolation artifacts they suffer. TMT complement (TMTc) ions, consisting of the peptide plus TMT balancer moieties, have the potential to provide quantitative read-outs essentially free of co-isolation artifacts, so long as they can be sufficiently resolved.Hendrik Wesseling; Konstantin Aizikov; Dmitry Grinfeld; Arne Kreutzmann; Daniel Marc Mourad; Oliver Lange; Alexander Makarov; Erik Hett; David H. PerlmanMerck Research Labs Cambridge Exploratory Science Center; Thermo Fisher Scientific (Bremen)
WP 028 – Ultrafast bacterial detection in Urine using high-throughput proteomics and artificial intelligenceAccording to the World Health Organization, antibiotic resistance is one of the biggest threats to global health, food security, and development today. To limit the emergence and spreading of new resistances, fast diagnostic tools needs to be developed in order to avoid the overprescription of broad-spectrum antibiotics. Low cost and fast MALDI-TOF MS technology has become an approach of choice for bacterial identification. However, it has several drawbacks: it requires a long step of bacterial culture prior to analysis (>24h), it has a low specificity and is not quantitative.Florence Roux; Mickaël Leclercq; Nicolai Bache; Tabiwang N. Arrey; Clarisse Gotti-Barban; Dorte B. Bekker-Jensen; Simon Pelletier; Maurice Boissinot; Bernard Delanghe; Claire Dauly; Michel G. Bergeron; Arnaud DroitCHU de Québec - Université Laval Research Center; Evosep Biosystems; Thermo Fisher Scientific
Validation of an Automated Platform for In Cell Footprinting StudiesThe footprinting method Fast Photochemical Oxidation of proteins (FPOP) has been extended to live cells in a method entitled in-cell FPOP (IC-FPOP). Owing to microsecond time scale of labeling, IC-FPOP is suited to study fast biological processes in the complex cellular environment. While a single cell flow system laid the foundation for the success of IC-FPOP, its temporal limitation motivated the design of a higher throughput platform, Platform Incubator with Movable XY stage (PIXY). Advancements have been made to automate the PIXY system. Currently, validation studies strive to address the utility of automation for IC-FPOP. This current study validates PIXY’s ability to accommodate automated time points and subsequent changes over time targeting the endoplasmic reticulum stress pathway.Dante T Johnson; Benjamin Punshon Smith; Lisa M. JonesUniversity of Maryland Baltimore School of Pharmacy; University of Maryland Baltimore County
FP 110 – Leveraging a Higher Duty Cycle DIA Acquisition On a Novel QTOF for Enhanced Proteomics AnalysisThe ability to identify and quantify large number of proteins and peptides is of great importance in translational medicine and life science research. Data independent acquisition (DIA) approaches have been shown to surpass data dependent acquisition (DDA) methodologies in terms of protein identifications in complex matrices especially at shorter acquisition speeds. Our new QTOF system equipped with a novel Zeno trap is able to deliver sensitivity gains in variable window SWATH acquisition. The built-in Zeno trap increases duty cycle at MS/MS level to over 90%, allowing for unprecedented gains in sensitivity (5-20x) at MS/MS, resulting in more identifications using Zeno SWATH acquisition. We evaluated increases in protein and peptide identifications using Zeno SWATH vs. SWATH acquisitions at various sample throughputs.Ihor Batruch; Yves Leblanc; Jason Causon; Naomi Diaz; Tatjana Talamantes; Anjali Chelur; Nic G. Bloomfield; Stephen Tate; Jose Castro-PerezSCIEX, Concord; SCIEX, Framingham
MP 162 – Combination of library search and database search on DIA dataSince Venable et al. first introduced data-independent acquisition (DIA) in 2004, DIA acquisition and data analysis tools have been continuously improved, making DIA a vital technology to identify and quantify thousands of proteins with high reproducibility and deep proteomics coverage. DIA data analysis, in general, relies on a spectral library constructed from data-dependent acquisition (DDA). Alternatively, the library-free approach searches DIA data directly against a fasta database. We combined a recently developed CCS-aware ProLuCID-4D search engine using ion mobility and a spectral library-based DIA approach to increase coverage.Robin Park; Qin Fu; Tharan Srikumar; Michael Krawitzky; Chistopher Adams; Dennis Trede; Gary Kruppa; Jennifer E. Van Eyk; Rohan ThakurBruker; Cedars-Sinai Medical Center; Bruker Ltd; Bruker, Inc.; Bruker Daltonics
WP 289 – Detection of SARS-CoV-2 antigen and its variants from clinical specimens using mass spectrometry-based targeted assaysThe COVID-19 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has overwhelmed health systems worldwide and highlighted limitations of diagnostic testing. Several types of diagnostic tests including RT-PCR-based assays and antigen detection by lateral flow assays, each with their own strengths and weaknesses, have been developed and deployed in short time. Recent emergence of new SARS-CoV-2 variants has necessitated modification of current tests requiring additional test development efforts. Detection of viral antigen using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a potential approach for detecting viral antigens and their variants. Here, we describe an immunoaffinity purification approach followed a by high resolution mass spectrometry-based targeted qualitative assay capable of detecting SARS-CoV-2 viral antigens and their variants from nasopharyngeal swab samples.Santosh Renuse; Anthony D. Maus; Patrick M. Vanderboom; Joseph Blommel; Jennifer V. Kemp; Kari M. Gurtner; Calvin Jerde; Anil K. Madugundu; Sandip Chavan; Jane A. Peterson; Benjamin J. Madden; Kiran Kumar Mangalaparthi; Dong-Gi Mun; Smrita SinghMayo Clinic; Institute of Bioinformatics, Bengaluru; Manipal Academy of Higher Education; National Institute of Mental Health and Neurosciences, Bengaluru
TOG pm – A complete and automated sample preparation strategy for high throughput and standardized proteomicsMass spectrometry-based proteomics is fast growing and provides a powerful set of technologies, with the potential to revolutionize health care and enable precision medicine. The Evosep One is specifically designed for high throughput applications, with a focus on clinical analysis of large sample cohorts.For a widespread adoption of proteomics in the clinic, the entire workflow from sample preparation through LC-MS and data analysis needs to be fast and robust. To meet these requirements, we introduce an end-to-end workflow from protein lysate to peptides loaded on Evotips ready for injection on the Evosep One. Furthermore to facilitate high throughput analysis in the clinical laboratory, we have developed a new standardized method the Evosep One allowing 500 samples analyzed per day.Dorte B. Bekker-Jensen; Florian S. Harking; Angela Mcardle; Lasse Falkenby; Jakob Bunkenborg; Ole B. Hørning; Jesper V Olsen; Nicolai BacheEvosep Biosystems; University of Copenhagen
MP 176 – Inference of Collisional Cross-Sections of peptides in an Orbitrap Mass AnalyzerRecently, the development of mass spectrometers coupled to ion-mobility devices have facilitated the accurate measurement of peptide collisional cross sections (CCS) adding an extra dimension to proteomics investigations. Recently, a method that do not require a dedicated ion mobility cell was described for determining CCS values of protein ions based on their decay rates in the time-domain transient signal in an Orbitrap mass analyzer. Here we present an extension of this strategy to peptide CCS inference in complex proteomics samples by introducing a minor hardware modification to an Orbitrap Exploris 480 mass spectrometer. We benchmarked the performance of this approach by analyzing HeLa lysates comparing to previously published CCS values from ion-mobility datasets showing comparable results.Ulises H Guzmán; Kyle Fort; Konstantin Aizikov; Martin Rykaer; Jeppe Madsen; Ana Martínez-Val; Alexander Makarov; Jesper OlsenNovo Nordisk Foundation Center for Protein Research; Thermo Fisher Scientific (Bremen) GmbH
ThOE am: Pre-formed gradients for high-throughput and robust nanoflow lipidomics with trapped ion mobility and PASEFLiquid chromatography – mass spectrometry (LC-MS)-based lipidomics is almost exclusively performed with analytical flow rates due to its throughput and robust operation. However, higher flow rates and larger column diameters lower the electrospray efficiency and hence sensitivity. To increase throughput and robustness of nanoflow separation, the Evosep One LC system elutes samples from disposable trap columns and embeds the analytes in a pre-formed gradient, which is then pushed through the separation column with a single high-pressure pump (PMID: 30104208). Here, we adapt this system to the specific challenges of lipidomics and evaluate its performance in combination with trapped ion mobility spectrometry (TIMS) and deep MS/MS analysis in single runs by parallel accumulation – serial fragmentation (PASEF, PMID: 31949144).Catherine G. Vasilopoulou; Nicolai Bache; Ole B. Hoerning; Fabrizio Merciai; Patricia Skowronek; Philipp E. Geyer; Karolina Sulek; Andreas-David Brunner; Aiko Barsch; Matthias Mann; Florian MeierMax Planck Institute of Biochemistry; Evosep Biosystemsk; University of Salerno; NNF Center for Protein Research ; Bruker Daltonics GmbH & Co. KG; Jena University Hospital
FP 706 – Label-free quantification of oxidized peptides in eHAP cell lines via a high-throughput dia-PASEF workflowdia-PASEF combines the advantages of data-independent acquisition (DIA), such as reproducible and accurate protein identification and quantification, with the benefits of ion mobility as additional separation dimension for the high-throughput analysis of complex proteomics samples. The ion mobility information can be used for alignment of precursor and fragment spectra, leading to higher confidence in protein identification. Herein, we apply the dia-PASEF workflow in combination with an Evosep One (Evosep) chromatography system for the high-throughput analysis of eHAP cell line digests, while quantifying spiked-in, oxidized target peptides in Spectronaut TM (Biognosys) on different gradient lengths.Romano Hebeler; Renata Blatnik; Philipp Strohmidel; Hamish Pegg; Joanna KirkpatrickBruker Daltonics GmbH & Co. KG; The Francis Crick Institute
MP 001- SPIN – Species by Proteome INvestigationGenetic species determination has become an indispensable tool in forensics, archaeology, and ecology. However, current technology requires a tradeoff between speed and versatility. Fast and cost-efficient methods like PCR or immunoassays can only detect single taxa, whereas global species identification by DNA or protein sequencing requires more time and resources. Although a good compromise was found with collagen-based peptide mass fingerprinting, the approach is not broadly used due a lack of accessible data interpretation software, missing confidence of fragment-based peptide identification, and the low variability of collagen sequences between close species. Here, we introduce “Species by Proteome INvestigation” (SPIN), a fast and automated proteomics workflow capable of querying over 150 mammalian species in 7.2 minutes of mass spectrometry (MS) analysis.Patrick Leopold Rüther; Immanuel M Husic; Pernille Bangsgaard; Kristian M Gregersen; Pernille Pantmann; Milena Carvalho; Ricardo M Godinho; Lukas Friedl; João Cascalheira; Marie Louise S Jørkov; Michael M Benedetti;NNF Center for Protein Research; Leipniz-Forschungsinstitut fuer Molekulare Pharmakologie; Globe Institute; Institute of Conservation; Natural History Museum of Denmark; Archaeology Dept., Museum Nordsjaelland
MP 257 – LiDIA-PASEF: A framework using experimental libraries for optimal acquisition of (phospho)proteomes by diaPASEFIn modern mass spectrometry (MS)-based proteomics, high acquisition speed, depth, quantitative accuracy, and data completeness across many runs are the key to reconstructing signaling pathways and protein expression profiles in health and disease. Combining data-independent acquisition (DIA) with parallel accumulation – serial fragmentation (PASEF), diaPASEF uses the correlation between mass-to-charge (m/z) and ion mobility (IM) in trapped ion mobility mass spectrometry (TIMS) and has shown great promise for high throughput applications. However, so far, the implicit trade-off between variable window width, cycle time, and diaPASEF windows placement in the two-dimensional m/z to IM space on the depth and quantitative accuracy has not been addressed. Here, we explore library-based diaPASEF methods (LiDIA-PASEF) applied to high throughput (phospho-)proteomics.Patricia Skowronek; Marvin Thielert; Fynn M. Hansen; Eugenia Voytik; Özge Karayel; Maria C. Tanzer; Florian Meier; Andreas-David Brunner; Matthias MannMPI; Jena University Hospital; NNF Center for protein research, Copenhagen
FP 112 – Using state of the art data independent acquisition (DIA) methods for protein identification in complex mixturesData dependent acquisition (DDA) methods have been the workhorse of protein/peptide identification by mass spectrometry, however, the stochastic ion selection process creates randomness that has been discussed numerous times. Also, as proteomic matrices are so complex, there is a proportion of MS/MS spectra with co-isolated precursor ions which is very difficult to deconvolute and can create identification confidence issues.Data independent acquisition (DIA) methods provide a route to deconvolute the MS/MS and generate a more robust and reproducible compound identification list. This paper describes DIA methods that provide excellent reproducible protein lists from samples which far improve on the reproducibility of DDA and also far increase our depth of coverage in a single proteomic sample.Stephen Tate; Yves LeblancSCIEX
MP 039 – Rapid proteome analysis using DIA and super-resolution Orbitrap mass spectrometryWith increasing focus on large-scale mass spectrometry studies, especially in clinical proteomics, there is a growing need for robust and sensitive high-throughput methods. Recent technological advances, such as preformed gradients in the Evosep One LC system, allow for the analysis of up to three hundred samples per day. Although appealing for high-throughput applications, these short gradients provide less coverage of the proteome and necessitate shorter cycle times in data-independent acquisition (DIA) strategies. Although this can be accommodated by increasing spectral complexity (larger DIA windows) or lowering mass resolution, data quality is reduced. To counteract this, we implement the Phase-Constrained Spectrum Deconvolution Method (ΦSDM) for Orbitrap signal processing, increasing acquisition speed for improved spectra quality and protein identification in short gradients.Sophia Steigerwald; Lili Niu; Kyle Fort; Arne Kreutzmann; Daniel Marc Mourad; Konstantin Aizikov; Dmitry Grinfeld; Alexander Makarov; Florian Meier; Matthias MannMax-Planck Institute of Biochemistry; Novo Nordisk Foundation Center for Protein Research – University of Copenhagen; Thermo Fisher Scientific (Bremen) GmbH; University Hospital Jena
FP 686 – Advantages of a dynamic polygon for MHC class I and II immunopeptidesMHC-associated peptides powerfully modulate T cell immunity and play a critical role in generating effective anti-tumor immune responses. Characterization of these peptides helps to generate therapeutic treatments and gain information on T cell mediated biomarkers. These peptides are challenging to characterize due to similar length, sequence conservation and lacking defined termini when compared to peptides generated upon enzymatic digestion. To overcome these challenges, use of PASEF (Parallel Accumulation and Serial Fragmentation) enables to generate high quality peptide spectra and resolve coeluting and isobaric peptides. Moreover, the capability to easily tailor the mobility space enables preferential detection of groups and sub-groups of relevant peptides.Francesco Pingitore; Michael Krawitzky; Josh Elias; Christopher AdamsBruker Daltonics; Chan Zuckerberg Biohub
FP 425 – Ultra-sensitive proteome quantification on the timsTOF SCP mass spectrometerSingle cell proteomics is a relatively young niche of proteomics compared to single cell genomics. In the recent years, significant progress has been made in sample handling and boosting the sample signal by multiplexing with mass spectrometers. timsTOF SCP is the first commercially introduced mass spectrometer dedicated for single cell proteomics. The modified front-end (orthogonal ion-guiding) of the instrument increase the ion transfer up to five times and keeps ultra-high robustness – the default attribute of the timsTOF platform. Here we demonstrate the performance of the instrument for low sample loads in the range of 250 pg to 1 ng in combination with robust low flow rate delivery from the Evosep system.Kristina Marx; Renata Blatnik; Verena Tellström; Markus Lubeck; Oliver Raether; Nagarjuna Nagaraj; Gary KruppaBruker Daltonics GmbH & Co.KG
WP 171 – Implementation of Data-independent Acquisition Parallel Accumulation-serial Fragmentation (dia-PASEF) to Investigate Accurate and Quantitative Biomarker Discovery in Human Myocardia-Demand IschemiaTrapped Ion Mobility Spectrometry (TIMS) separates ions based on their mobility across an electric field and with subsequent release based on gas phase mobility. Parallel accumulation serial fragmentation (PASEF) synchronizes TIMS with MS/MS precursor selection allowing fragmentation of multiple precursors per scan. dia-PASEF-MS is reported to have higher sensitivity and quantitative accuracy. As an applied bioanalytical tool in clinical research, MS based biomarker discovery involves quantifying hundreds of plasma proteins. Here, we present a robust workflow combining dia-PASEF-MS with a standardized liquid chromatography platform on a timsTOF for quantitative and accurate biomarker discovery.After implementation of a robust dia-PASEF workflow, we identified candidate markers of myocardial-demand ischemia for earlier therapeutic intervention in patients with clinical suspicion of a heart attack.Qin Fu; Michael Krawitzk; Kirstin Washington; Simion Kreimer; Chistopher Adams; Gary Kruppa; Jennifer E. Van EykCedars-Sinai Medical Center; Bruker Daltonics
FP 265 – DeGlyPHER: an ultrasensitive method for analysis of viral spike N-glycoformsViruses can evade the host immune system by displaying numerous glycans on their surface ‘spike-proteins’ that cover immune epitopes. We have developed an ultrasensitive ‘single pot’ method to assess glycan occupancy and the extent of glycan processing from high-mannose to complex forms at each N-glycosylation site. Though aimed at characterizing glycosylation of viral spike-proteins as potential vaccines, this method is applicable for analysis of site-specific glycosylation of any glycoprotein.Sabyasachi Baboo; Jolene K. Diedrich; Salvador Martinez-Bartolome; Xiaoning Wang; Torben Schiffner; Bettina Groschel; William R. Schief; James C. Paulson; John R. Yates, IIIThe Scripps Research Institute; IAVI Neutralizing Antibody Center; The Ragon Institute of Massachusetts General Hospital
ThP 006 – New LC-MS Platforms for Primary and High-Order Structural Characterization of a Biotherapeutic Immunocaptured from SerumCharacterization of complex biotherapeutics to assess susceptibilities and product quality attributes is routinely performed during biopharmaceutical product development, but characterization of material from serum offers a physiological perspective and may predict drug performance in vivo. In this work, a non-covalent antibody-small molecule (mAb-NCE) complex from rat serum was screened for stability using LC-MS approaches. Quality attribute monitoring of immunocaptured antibody was performed for tryptic peptides containing Met, Asn, Asp or Lys residues from the CDR/variable regions for oxidation, deamidation, isomerization and glycation quantitation, respectively. In addition, a native protein MS method was developed to characterize intact mAb-NCE complex from serum. LC-MS data enabled ex vivo stability assessment of sequence-related modifications and may be applied to determine stoichiometric ratio of mAb/ligand.Nicole A. Schneck; Timothy W. Sikorski; John T. Mehl; John F. KellieGSK
ThOB pm: Biomarkers: Quantitative Analysis – PRM-Studio: Improved data extraction and quality control of peptide PRM data for complex clinical samplesMass spectrometry–based targeted proteomics allows objective protein quantitation of clinical biomarkers, including from formalin-fixed, paraffin-embedded (FFPE) human tumor biopsies. Each tumor biopsy is a distinct complex matrix, with potential for interference with the target peptide fragment ions. The combination of stable-isotope labelled internal standard (SIS) peptides and high-resolution detection in a parallel reaction monitoring experiment mitigates the interference problem. We deploy a novel data analysis pipeline allowing: 1) narrow extraction windows in the retention time dimension; 2) reporting the differences of the deviations from the expected m/z values, between SIS and target peptide fragment ions to flag possible interference. We highlight the benefits of this approach compared to the widely used Skyline platform for complex FFPE tissue sample sets.Steve M Sweet; Wen Yu; Yeoun Jin KimAstraZeneca
WP 299 – High Throughput Plasma Glycated Proteome Profiling to Discover Cardiovascular Disease Biomarkers from the Epidemiology of Diabetes Interventions and Complications StudyThe Epidemiology of Diabetes Interventions and Complications (EDIC) study was an observational follow-up of the Diabetes Control and Complications Trial (DCCT) to examine the long-term effects of the original DCCT interventions on diabetic complications such as cardiovascular diseases (CVD). Current observational based prediction models are inadequate in prediction of CVD. Amadori products originating from early step of protein glycation are associated with long-term complications. The aim of this study is to investigate whether Amadori products are sensitive molecular biomarkers for early diagnosis of CVD in the type 1 diabetes (T1D) population. To do so, we used high throughput and robust boronate affinity enrichment-based proteomics to profile the glycated proteins from the plasma samples collected in the EDIC study.Yang Tang; Laura Pyle; Timothy Vigers; Randi Johnson; Janet Snell-Bergeon; Qibin ZhangCenter for Translational Biomedical Research; College of Medicine, University of Colorado Anschutz Medical campus; Department of Chemistry & Biochemistry, University of North Carolina
FP 101 – Evaluation of LC-MS/MS Instrumentation for Targeted Analysis of SARS-CoV-2 PeptidesSince the outbreak of the global COVID-19 pandemic, the potential for liquid chromatography- tandem mass spectrometry (LC-MS/MS) analyses of SARS-CoV-2 peptides to provide complementary and supplemental testing to established quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays has been extensively explored. However, matching the throughput and sensitivity of qRT-PCR has proven challenging. High performance instrumentation and low flow rates are necessary for detection of positive samples with low viral loads, but lower flow rates generally result in longer analysis times. An additional consideration that is often overlooked in publications discussing LC-MS/MS analyses of SARS-CoV-2 is carry-over of peptides from samples with high viral loads. In this work, we describe our evaluation of numerous platforms for optimal detection of SARS-CoV-2 peptides.Anthony Maus; Santosh Renuse; Jennifer Kemp; Ravinder J. Singh; Akhilesh Pandey; Stefan K. GrebeMayo Clinic
MP 024 – Evaluation of dia-PASEF for short gradient using library and library free approachesdia-PASEF (Meier et.al.,2019) takes advantage of the additional dimension of separation provided by trapped ion mobility (TIMS) for the analysis of complex proteomics samples by data independent analysis (DIA). The CCS (Collisional Cross Section) value determined for each precursor can also be used for alignment of precursor and fragment information. ­­ Here, we evaluate benefits of dia-PASEF comparing very short and long gradients. Moreover, we compared results from two independent software platforms (Spectronaut 15 and DIA-NN 1.8) which can process native dia-PASEF data using spectral libraries or a library-free approach.Diego M Assis; Elizabeth Gordon; Matthew WillettsBruker Scientific, LLC
FP 111 – In-depth proteome analysis of bronchoalveolar lavage fluid from a chronic obstructive pulmonary disease model using high throughput 4D-diaPASEFBronchoalveolar lavage fluid (BALF) is a biological fluid that is comprised of airway epithelial, immune cells, and soluble materials. It reflects the cellular and molecular states of the lung and can provide critical insights into pathogenesis of pulmonary diseases as well as valuable biomarker candidates. However, the BALF proteome is dominated by plasma-derived high-abundance proteins that lead to a large dynamic range creating a challenge for comprehensive profiling. Herein we address this challenge by developing a rapid four-dimension LC-MS approach using trapped ion mobility spectrometry (TIMS) and parallel accumulation serial fragmentation (PASEF) with a data-independent acquisition (DIA) method. Our workflow facilitates high-throughput capability that achieves deep proteome coverage and high quantitative reproducibility for BALF analysis.Abby Chiang; Chelsea C Boo; Raghothama Chaerkady; Tara Kenny; John Kim; Antonio Digiandomenico; Lisa Cazares; Sonja HessAstraZeneca
MP 038 – Achieving high performance with EvoSep One using optimized analytical column configurations for Data-Independent Acquisition (DIA) using the Bruker timsTOF-MS instrumentData-independent acquisition (DIA) strategies coupled with Evosep chromatography have recently gained popularity in achieving a high-throughput and robust mass spectrometry-based proteomics, but with compromised peptide identifications leading to lower peptide to protein ratios. Since premade gradient methods fix the flow rate and gradient length in the Evosep, a limited number of analytical columns fitting pressure/flow profiles can be used for peptide separation. To circumvent this limitation, we evaluated and standardized the performance of different dimensions of analytical columns with the Evosep and compared it with an EasyLC using a timsTOF-MS instrument in diaPASEF mode. The results reveal optimized usage of a specific column dimension that achieves higher sensitivity and deep proteome coverageMukul K. Midha; Timm J. Prokop; Charu Kapil; Peter A. Nielsen; Robert L. MoritzInstitute for Systems Biology, PepSep
TitleSubjectMaterialYearSummaryAuthorsOrganizationEvosep methodMS instrumentationLearn Moredoc_tag_hfilterevosep_method_hfilterms_vendor_hfilter
TP 021 – Deep and high-throughput urine proteomics profiling for the classification of Parkinson’s disease patientsParkinson’s disease (PD) is the second most abundant neurodegenerative disease (after Alzheimer’s disease) with a prevalence of about 1% for over 60-year-olds. The Parkinson’s Progression Markers Initiative (PPMI) is an observational clinical study for the monitoring of PD progression and identification of new biomarkers. It comprises about 1500 patients in the initial cohort and close to 5000 patients scheduled for an expansion of the study. Previous studies using smaller cohorts have shown that urinary proteome profiling is a promising strategy for the detection of novel biomarkers and patient stratification. We employed our automated high-throughput mass spectrometry (MS)-based proteomics pipeline to the PPMI cohort to identify novel biomarkers for PD.Johannes B Mueller-Reif; Philipp E. Geyer; Sophia Doll; Felix M Riese; Shalini Padmanabhan; Kalpana Merchant; Magnus Schwoerer; Sebastian Virreira WinterOmicEra Diagnostics GmbH; The Michael J. Fox Foundation for Parkinson’s Research; Northwestern University Feinberg School of Medicine
MP 035 – Opportunities and challenges for scaling quantitative proteomics studies with high-throughput DIA-MSQuantitative mass spectrometry is increasingly being used in high-throughput proteomics studies, with ever increasing cohorts and sample sizes. These studies are in part enabled by data independent acquisition (DIA/SWATH) to systematically measure peptides by label-free quantification (LFQ), which gives comprehensive peptide detection and quantification at theoretically unlimited throughput. However, there is limited literature detailing the quantitative challenges of performing proteomics at 100s of samples per week, with most studies focused on the number of detections which are reported. We set out to deeply characterize a high-throughput DIA-MS workflow and assess, empirically, whether accounting for technical variance with quality control and system suitability can be used to improve the accuracy and precision of quantitative proteomics experiments.Lindsay K Pino; Rico Meinl; Erin Broderick; Noelle Schumacker; Daniele Canzani; William E Fondrie; Alexander FederationTalus Bioscience
WP 330 – Beyond TurboTMT: Phi-SDM Super-resolution Methods for Next-generation Highly-multiplexed Quantitative Ultrasensitive and Single-Cell Proteomics via TMTPro Complement Ion DeconvolutionSingle-cell Proteomics has emerged as an extremely promising new field capable of unlocking the vast cellular heterogeneity that defines biology, disease, and therapeutic response. However, it presents enormous challenges, including maximizing both quantitative signal and experimental throughput. We utilize the multiplexed method (e.g., SCOPE-MS/SCOPE2) in which individual cells’ proteomes are labeled, pooled, and combined with abundant carrier material using TMT tags, improving both peptide identification and cell throughput. Still, accurate quantitation via TMT reporter ions remains challenging, in part due to the co-isolation artifacts they suffer. TMT complement (TMTc) ions, consisting of the peptide plus TMT balancer moieties, have the potential to provide quantitative read-outs essentially free of co-isolation artifacts, so long as they can be sufficiently resolved.Hendrik Wesseling; Konstantin Aizikov; Dmitry Grinfeld; Arne Kreutzmann; Daniel Marc Mourad; Oliver Lange; Alexander Makarov; Erik Hett; David H. PerlmanMerck Research Labs Cambridge Exploratory Science Center; Thermo Fisher Scientific (Bremen)
WP 028 – Ultrafast bacterial detection in Urine using high-throughput proteomics and artificial intelligenceAccording to the World Health Organization, antibiotic resistance is one of the biggest threats to global health, food security, and development today. To limit the emergence and spreading of new resistances, fast diagnostic tools needs to be developed in order to avoid the overprescription of broad-spectrum antibiotics. Low cost and fast MALDI-TOF MS technology has become an approach of choice for bacterial identification. However, it has several drawbacks: it requires a long step of bacterial culture prior to analysis (>24h), it has a low specificity and is not quantitative.Florence Roux; Mickaël Leclercq; Nicolai Bache; Tabiwang N. Arrey; Clarisse Gotti-Barban; Dorte B. Bekker-Jensen; Simon Pelletier; Maurice Boissinot; Bernard Delanghe; Claire Dauly; Michel G. Bergeron; Arnaud DroitCHU de Québec - Université Laval Research Center; Evosep Biosystems; Thermo Fisher Scientific
Validation of an Automated Platform for In Cell Footprinting StudiesThe footprinting method Fast Photochemical Oxidation of proteins (FPOP) has been extended to live cells in a method entitled in-cell FPOP (IC-FPOP). Owing to microsecond time scale of labeling, IC-FPOP is suited to study fast biological processes in the complex cellular environment. While a single cell flow system laid the foundation for the success of IC-FPOP, its temporal limitation motivated the design of a higher throughput platform, Platform Incubator with Movable XY stage (PIXY). Advancements have been made to automate the PIXY system. Currently, validation studies strive to address the utility of automation for IC-FPOP. This current study validates PIXY’s ability to accommodate automated time points and subsequent changes over time targeting the endoplasmic reticulum stress pathway.Dante T Johnson; Benjamin Punshon Smith; Lisa M. JonesUniversity of Maryland Baltimore School of Pharmacy; University of Maryland Baltimore County
FP 110 – Leveraging a Higher Duty Cycle DIA Acquisition On a Novel QTOF for Enhanced Proteomics AnalysisThe ability to identify and quantify large number of proteins and peptides is of great importance in translational medicine and life science research. Data independent acquisition (DIA) approaches have been shown to surpass data dependent acquisition (DDA) methodologies in terms of protein identifications in complex matrices especially at shorter acquisition speeds. Our new QTOF system equipped with a novel Zeno trap is able to deliver sensitivity gains in variable window SWATH acquisition. The built-in Zeno trap increases duty cycle at MS/MS level to over 90%, allowing for unprecedented gains in sensitivity (5-20x) at MS/MS, resulting in more identifications using Zeno SWATH acquisition. We evaluated increases in protein and peptide identifications using Zeno SWATH vs. SWATH acquisitions at various sample throughputs.Ihor Batruch; Yves Leblanc; Jason Causon; Naomi Diaz; Tatjana Talamantes; Anjali Chelur; Nic G. Bloomfield; Stephen Tate; Jose Castro-PerezSCIEX, Concord; SCIEX, Framingham
MP 162 – Combination of library search and database search on DIA dataSince Venable et al. first introduced data-independent acquisition (DIA) in 2004, DIA acquisition and data analysis tools have been continuously improved, making DIA a vital technology to identify and quantify thousands of proteins with high reproducibility and deep proteomics coverage. DIA data analysis, in general, relies on a spectral library constructed from data-dependent acquisition (DDA). Alternatively, the library-free approach searches DIA data directly against a fasta database. We combined a recently developed CCS-aware ProLuCID-4D search engine using ion mobility and a spectral library-based DIA approach to increase coverage.Robin Park; Qin Fu; Tharan Srikumar; Michael Krawitzky; Chistopher Adams; Dennis Trede; Gary Kruppa; Jennifer E. Van Eyk; Rohan ThakurBruker; Cedars-Sinai Medical Center; Bruker Ltd; Bruker, Inc.; Bruker Daltonics
WP 289 – Detection of SARS-CoV-2 antigen and its variants from clinical specimens using mass spectrometry-based targeted assaysThe COVID-19 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has overwhelmed health systems worldwide and highlighted limitations of diagnostic testing. Several types of diagnostic tests including RT-PCR-based assays and antigen detection by lateral flow assays, each with their own strengths and weaknesses, have been developed and deployed in short time. Recent emergence of new SARS-CoV-2 variants has necessitated modification of current tests requiring additional test development efforts. Detection of viral antigen using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a potential approach for detecting viral antigens and their variants. Here, we describe an immunoaffinity purification approach followed a by high resolution mass spectrometry-based targeted qualitative assay capable of detecting SARS-CoV-2 viral antigens and their variants from nasopharyngeal swab samples.Santosh Renuse; Anthony D. Maus; Patrick M. Vanderboom; Joseph Blommel; Jennifer V. Kemp; Kari M. Gurtner; Calvin Jerde; Anil K. Madugundu; Sandip Chavan; Jane A. Peterson; Benjamin J. Madden; Kiran Kumar Mangalaparthi; Dong-Gi Mun; Smrita SinghMayo Clinic; Institute of Bioinformatics, Bengaluru; Manipal Academy of Higher Education; National Institute of Mental Health and Neurosciences, Bengaluru
TOG pm – A complete and automated sample preparation strategy for high throughput and standardized proteomicsMass spectrometry-based proteomics is fast growing and provides a powerful set of technologies, with the potential to revolutionize health care and enable precision medicine. The Evosep One is specifically designed for high throughput applications, with a focus on clinical analysis of large sample cohorts.For a widespread adoption of proteomics in the clinic, the entire workflow from sample preparation through LC-MS and data analysis needs to be fast and robust. To meet these requirements, we introduce an end-to-end workflow from protein lysate to peptides loaded on Evotips ready for injection on the Evosep One. Furthermore to facilitate high throughput analysis in the clinical laboratory, we have developed a new standardized method the Evosep One allowing 500 samples analyzed per day.Dorte B. Bekker-Jensen; Florian S. Harking; Angela Mcardle; Lasse Falkenby; Jakob Bunkenborg; Ole B. Hørning; Jesper V Olsen; Nicolai BacheEvosep Biosystems; University of Copenhagen
MP 176 – Inference of Collisional Cross-Sections of peptides in an Orbitrap Mass AnalyzerRecently, the development of mass spectrometers coupled to ion-mobility devices have facilitated the accurate measurement of peptide collisional cross sections (CCS) adding an extra dimension to proteomics investigations. Recently, a method that do not require a dedicated ion mobility cell was described for determining CCS values of protein ions based on their decay rates in the time-domain transient signal in an Orbitrap mass analyzer. Here we present an extension of this strategy to peptide CCS inference in complex proteomics samples by introducing a minor hardware modification to an Orbitrap Exploris 480 mass spectrometer. We benchmarked the performance of this approach by analyzing HeLa lysates comparing to previously published CCS values from ion-mobility datasets showing comparable results.Ulises H Guzmán; Kyle Fort; Konstantin Aizikov; Martin Rykaer; Jeppe Madsen; Ana Martínez-Val; Alexander Makarov; Jesper OlsenNovo Nordisk Foundation Center for Protein Research; Thermo Fisher Scientific (Bremen) GmbH
ThOE am: Pre-formed gradients for high-throughput and robust nanoflow lipidomics with trapped ion mobility and PASEFLiquid chromatography – mass spectrometry (LC-MS)-based lipidomics is almost exclusively performed with analytical flow rates due to its throughput and robust operation. However, higher flow rates and larger column diameters lower the electrospray efficiency and hence sensitivity. To increase throughput and robustness of nanoflow separation, the Evosep One LC system elutes samples from disposable trap columns and embeds the analytes in a pre-formed gradient, which is then pushed through the separation column with a single high-pressure pump (PMID: 30104208). Here, we adapt this system to the specific challenges of lipidomics and evaluate its performance in combination with trapped ion mobility spectrometry (TIMS) and deep MS/MS analysis in single runs by parallel accumulation – serial fragmentation (PASEF, PMID: 31949144).Catherine G. Vasilopoulou; Nicolai Bache; Ole B. Hoerning; Fabrizio Merciai; Patricia Skowronek; Philipp E. Geyer; Karolina Sulek; Andreas-David Brunner; Aiko Barsch; Matthias Mann; Florian MeierMax Planck Institute of Biochemistry; Evosep Biosystemsk; University of Salerno; NNF Center for Protein Research ; Bruker Daltonics GmbH & Co. KG; Jena University Hospital
FP 706 – Label-free quantification of oxidized peptides in eHAP cell lines via a high-throughput dia-PASEF workflowdia-PASEF combines the advantages of data-independent acquisition (DIA), such as reproducible and accurate protein identification and quantification, with the benefits of ion mobility as additional separation dimension for the high-throughput analysis of complex proteomics samples. The ion mobility information can be used for alignment of precursor and fragment spectra, leading to higher confidence in protein identification. Herein, we apply the dia-PASEF workflow in combination with an Evosep One (Evosep) chromatography system for the high-throughput analysis of eHAP cell line digests, while quantifying spiked-in, oxidized target peptides in Spectronaut TM (Biognosys) on different gradient lengths.Romano Hebeler; Renata Blatnik; Philipp Strohmidel; Hamish Pegg; Joanna KirkpatrickBruker Daltonics GmbH & Co. KG; The Francis Crick Institute
MP 001- SPIN – Species by Proteome INvestigationGenetic species determination has become an indispensable tool in forensics, archaeology, and ecology. However, current technology requires a tradeoff between speed and versatility. Fast and cost-efficient methods like PCR or immunoassays can only detect single taxa, whereas global species identification by DNA or protein sequencing requires more time and resources. Although a good compromise was found with collagen-based peptide mass fingerprinting, the approach is not broadly used due a lack of accessible data interpretation software, missing confidence of fragment-based peptide identification, and the low variability of collagen sequences between close species. Here, we introduce “Species by Proteome INvestigation” (SPIN), a fast and automated proteomics workflow capable of querying over 150 mammalian species in 7.2 minutes of mass spectrometry (MS) analysis.Patrick Leopold Rüther; Immanuel M Husic; Pernille Bangsgaard; Kristian M Gregersen; Pernille Pantmann; Milena Carvalho; Ricardo M Godinho; Lukas Friedl; João Cascalheira; Marie Louise S Jørkov; Michael M Benedetti;NNF Center for Protein Research; Leipniz-Forschungsinstitut fuer Molekulare Pharmakologie; Globe Institute; Institute of Conservation; Natural History Museum of Denmark; Archaeology Dept., Museum Nordsjaelland
MP 257 – LiDIA-PASEF: A framework using experimental libraries for optimal acquisition of (phospho)proteomes by diaPASEFIn modern mass spectrometry (MS)-based proteomics, high acquisition speed, depth, quantitative accuracy, and data completeness across many runs are the key to reconstructing signaling pathways and protein expression profiles in health and disease. Combining data-independent acquisition (DIA) with parallel accumulation – serial fragmentation (PASEF), diaPASEF uses the correlation between mass-to-charge (m/z) and ion mobility (IM) in trapped ion mobility mass spectrometry (TIMS) and has shown great promise for high throughput applications. However, so far, the implicit trade-off between variable window width, cycle time, and diaPASEF windows placement in the two-dimensional m/z to IM space on the depth and quantitative accuracy has not been addressed. Here, we explore library-based diaPASEF methods (LiDIA-PASEF) applied to high throughput (phospho-)proteomics.Patricia Skowronek; Marvin Thielert; Fynn M. Hansen; Eugenia Voytik; Özge Karayel; Maria C. Tanzer; Florian Meier; Andreas-David Brunner; Matthias MannMPI; Jena University Hospital; NNF Center for protein research, Copenhagen
FP 112 – Using state of the art data independent acquisition (DIA) methods for protein identification in complex mixturesData dependent acquisition (DDA) methods have been the workhorse of protein/peptide identification by mass spectrometry, however, the stochastic ion selection process creates randomness that has been discussed numerous times. Also, as proteomic matrices are so complex, there is a proportion of MS/MS spectra with co-isolated precursor ions which is very difficult to deconvolute and can create identification confidence issues.Data independent acquisition (DIA) methods provide a route to deconvolute the MS/MS and generate a more robust and reproducible compound identification list. This paper describes DIA methods that provide excellent reproducible protein lists from samples which far improve on the reproducibility of DDA and also far increase our depth of coverage in a single proteomic sample.Stephen Tate; Yves LeblancSCIEX
MP 039 – Rapid proteome analysis using DIA and super-resolution Orbitrap mass spectrometryWith increasing focus on large-scale mass spectrometry studies, especially in clinical proteomics, there is a growing need for robust and sensitive high-throughput methods. Recent technological advances, such as preformed gradients in the Evosep One LC system, allow for the analysis of up to three hundred samples per day. Although appealing for high-throughput applications, these short gradients provide less coverage of the proteome and necessitate shorter cycle times in data-independent acquisition (DIA) strategies. Although this can be accommodated by increasing spectral complexity (larger DIA windows) or lowering mass resolution, data quality is reduced. To counteract this, we implement the Phase-Constrained Spectrum Deconvolution Method (ΦSDM) for Orbitrap signal processing, increasing acquisition speed for improved spectra quality and protein identification in short gradients.Sophia Steigerwald; Lili Niu; Kyle Fort; Arne Kreutzmann; Daniel Marc Mourad; Konstantin Aizikov; Dmitry Grinfeld; Alexander Makarov; Florian Meier; Matthias MannMax-Planck Institute of Biochemistry; Novo Nordisk Foundation Center for Protein Research – University of Copenhagen; Thermo Fisher Scientific (Bremen) GmbH; University Hospital Jena
FP 686 – Advantages of a dynamic polygon for MHC class I and II immunopeptidesMHC-associated peptides powerfully modulate T cell immunity and play a critical role in generating effective anti-tumor immune responses. Characterization of these peptides helps to generate therapeutic treatments and gain information on T cell mediated biomarkers. These peptides are challenging to characterize due to similar length, sequence conservation and lacking defined termini when compared to peptides generated upon enzymatic digestion. To overcome these challenges, use of PASEF (Parallel Accumulation and Serial Fragmentation) enables to generate high quality peptide spectra and resolve coeluting and isobaric peptides. Moreover, the capability to easily tailor the mobility space enables preferential detection of groups and sub-groups of relevant peptides.Francesco Pingitore; Michael Krawitzky; Josh Elias; Christopher AdamsBruker Daltonics; Chan Zuckerberg Biohub
FP 425 – Ultra-sensitive proteome quantification on the timsTOF SCP mass spectrometerSingle cell proteomics is a relatively young niche of proteomics compared to single cell genomics. In the recent years, significant progress has been made in sample handling and boosting the sample signal by multiplexing with mass spectrometers. timsTOF SCP is the first commercially introduced mass spectrometer dedicated for single cell proteomics. The modified front-end (orthogonal ion-guiding) of the instrument increase the ion transfer up to five times and keeps ultra-high robustness – the default attribute of the timsTOF platform. Here we demonstrate the performance of the instrument for low sample loads in the range of 250 pg to 1 ng in combination with robust low flow rate delivery from the Evosep system.Kristina Marx; Renata Blatnik; Verena Tellström; Markus Lubeck; Oliver Raether; Nagarjuna Nagaraj; Gary KruppaBruker Daltonics GmbH & Co.KG
WP 171 – Implementation of Data-independent Acquisition Parallel Accumulation-serial Fragmentation (dia-PASEF) to Investigate Accurate and Quantitative Biomarker Discovery in Human Myocardia-Demand IschemiaTrapped Ion Mobility Spectrometry (TIMS) separates ions based on their mobility across an electric field and with subsequent release based on gas phase mobility. Parallel accumulation serial fragmentation (PASEF) synchronizes TIMS with MS/MS precursor selection allowing fragmentation of multiple precursors per scan. dia-PASEF-MS is reported to have higher sensitivity and quantitative accuracy. As an applied bioanalytical tool in clinical research, MS based biomarker discovery involves quantifying hundreds of plasma proteins. Here, we present a robust workflow combining dia-PASEF-MS with a standardized liquid chromatography platform on a timsTOF for quantitative and accurate biomarker discovery.After implementation of a robust dia-PASEF workflow, we identified candidate markers of myocardial-demand ischemia for earlier therapeutic intervention in patients with clinical suspicion of a heart attack.Qin Fu; Michael Krawitzk; Kirstin Washington; Simion Kreimer; Chistopher Adams; Gary Kruppa; Jennifer E. Van EykCedars-Sinai Medical Center; Bruker Daltonics
FP 265 – DeGlyPHER: an ultrasensitive method for analysis of viral spike N-glycoformsViruses can evade the host immune system by displaying numerous glycans on their surface ‘spike-proteins’ that cover immune epitopes. We have developed an ultrasensitive ‘single pot’ method to assess glycan occupancy and the extent of glycan processing from high-mannose to complex forms at each N-glycosylation site. Though aimed at characterizing glycosylation of viral spike-proteins as potential vaccines, this method is applicable for analysis of site-specific glycosylation of any glycoprotein.Sabyasachi Baboo; Jolene K. Diedrich; Salvador Martinez-Bartolome; Xiaoning Wang; Torben Schiffner; Bettina Groschel; William R. Schief; James C. Paulson; John R. Yates, IIIThe Scripps Research Institute; IAVI Neutralizing Antibody Center; The Ragon Institute of Massachusetts General Hospital
ThP 006 – New LC-MS Platforms for Primary and High-Order Structural Characterization of a Biotherapeutic Immunocaptured from SerumCharacterization of complex biotherapeutics to assess susceptibilities and product quality attributes is routinely performed during biopharmaceutical product development, but characterization of material from serum offers a physiological perspective and may predict drug performance in vivo. In this work, a non-covalent antibody-small molecule (mAb-NCE) complex from rat serum was screened for stability using LC-MS approaches. Quality attribute monitoring of immunocaptured antibody was performed for tryptic peptides containing Met, Asn, Asp or Lys residues from the CDR/variable regions for oxidation, deamidation, isomerization and glycation quantitation, respectively. In addition, a native protein MS method was developed to characterize intact mAb-NCE complex from serum. LC-MS data enabled ex vivo stability assessment of sequence-related modifications and may be applied to determine stoichiometric ratio of mAb/ligand.Nicole A. Schneck; Timothy W. Sikorski; John T. Mehl; John F. KellieGSK
ThOB pm: Biomarkers: Quantitative Analysis – PRM-Studio: Improved data extraction and quality control of peptide PRM data for complex clinical samplesMass spectrometry–based targeted proteomics allows objective protein quantitation of clinical biomarkers, including from formalin-fixed, paraffin-embedded (FFPE) human tumor biopsies. Each tumor biopsy is a distinct complex matrix, with potential for interference with the target peptide fragment ions. The combination of stable-isotope labelled internal standard (SIS) peptides and high-resolution detection in a parallel reaction monitoring experiment mitigates the interference problem. We deploy a novel data analysis pipeline allowing: 1) narrow extraction windows in the retention time dimension; 2) reporting the differences of the deviations from the expected m/z values, between SIS and target peptide fragment ions to flag possible interference. We highlight the benefits of this approach compared to the widely used Skyline platform for complex FFPE tissue sample sets.Steve M Sweet; Wen Yu; Yeoun Jin KimAstraZeneca
WP 299 – High Throughput Plasma Glycated Proteome Profiling to Discover Cardiovascular Disease Biomarkers from the Epidemiology of Diabetes Interventions and Complications StudyThe Epidemiology of Diabetes Interventions and Complications (EDIC) study was an observational follow-up of the Diabetes Control and Complications Trial (DCCT) to examine the long-term effects of the original DCCT interventions on diabetic complications such as cardiovascular diseases (CVD). Current observational based prediction models are inadequate in prediction of CVD. Amadori products originating from early step of protein glycation are associated with long-term complications. The aim of this study is to investigate whether Amadori products are sensitive molecular biomarkers for early diagnosis of CVD in the type 1 diabetes (T1D) population. To do so, we used high throughput and robust boronate affinity enrichment-based proteomics to profile the glycated proteins from the plasma samples collected in the EDIC study.Yang Tang; Laura Pyle; Timothy Vigers; Randi Johnson; Janet Snell-Bergeon; Qibin ZhangCenter for Translational Biomedical Research; College of Medicine, University of Colorado Anschutz Medical campus; Department of Chemistry & Biochemistry, University of North Carolina
FP 101 – Evaluation of LC-MS/MS Instrumentation for Targeted Analysis of SARS-CoV-2 PeptidesSince the outbreak of the global COVID-19 pandemic, the potential for liquid chromatography- tandem mass spectrometry (LC-MS/MS) analyses of SARS-CoV-2 peptides to provide complementary and supplemental testing to established quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays has been extensively explored. However, matching the throughput and sensitivity of qRT-PCR has proven challenging. High performance instrumentation and low flow rates are necessary for detection of positive samples with low viral loads, but lower flow rates generally result in longer analysis times. An additional consideration that is often overlooked in publications discussing LC-MS/MS analyses of SARS-CoV-2 is carry-over of peptides from samples with high viral loads. In this work, we describe our evaluation of numerous platforms for optimal detection of SARS-CoV-2 peptides.Anthony Maus; Santosh Renuse; Jennifer Kemp; Ravinder J. Singh; Akhilesh Pandey; Stefan K. GrebeMayo Clinic
MP 024 – Evaluation of dia-PASEF for short gradient using library and library free approachesdia-PASEF (Meier et.al.,2019) takes advantage of the additional dimension of separation provided by trapped ion mobility (TIMS) for the analysis of complex proteomics samples by data independent analysis (DIA). The CCS (Collisional Cross Section) value determined for each precursor can also be used for alignment of precursor and fragment information. ­­ Here, we evaluate benefits of dia-PASEF comparing very short and long gradients. Moreover, we compared results from two independent software platforms (Spectronaut 15 and DIA-NN 1.8) which can process native dia-PASEF data using spectral libraries or a library-free approach.Diego M Assis; Elizabeth Gordon; Matthew WillettsBruker Scientific, LLC
FP 111 – In-depth proteome analysis of bronchoalveolar lavage fluid from a chronic obstructive pulmonary disease model using high throughput 4D-diaPASEFBronchoalveolar lavage fluid (BALF) is a biological fluid that is comprised of airway epithelial, immune cells, and soluble materials. It reflects the cellular and molecular states of the lung and can provide critical insights into pathogenesis of pulmonary diseases as well as valuable biomarker candidates. However, the BALF proteome is dominated by plasma-derived high-abundance proteins that lead to a large dynamic range creating a challenge for comprehensive profiling. Herein we address this challenge by developing a rapid four-dimension LC-MS approach using trapped ion mobility spectrometry (TIMS) and parallel accumulation serial fragmentation (PASEF) with a data-independent acquisition (DIA) method. Our workflow facilitates high-throughput capability that achieves deep proteome coverage and high quantitative reproducibility for BALF analysis.Abby Chiang; Chelsea C Boo; Raghothama Chaerkady; Tara Kenny; John Kim; Antonio Digiandomenico; Lisa Cazares; Sonja HessAstraZeneca
MP 038 – Achieving high performance with EvoSep One using optimized analytical column configurations for Data-Independent Acquisition (DIA) using the Bruker timsTOF-MS instrumentData-independent acquisition (DIA) strategies coupled with Evosep chromatography have recently gained popularity in achieving a high-throughput and robust mass spectrometry-based proteomics, but with compromised peptide identifications leading to lower peptide to protein ratios. Since premade gradient methods fix the flow rate and gradient length in the Evosep, a limited number of analytical columns fitting pressure/flow profiles can be used for peptide separation. To circumvent this limitation, we evaluated and standardized the performance of different dimensions of analytical columns with the Evosep and compared it with an EasyLC using a timsTOF-MS instrument in diaPASEF mode. The results reveal optimized usage of a specific column dimension that achieves higher sensitivity and deep proteome coverageMukul K. Midha; Timm J. Prokop; Charu Kapil; Peter A. Nielsen; Robert L. MoritzInstitute for Systems Biology, PepSep

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Evosep at ASMS 2020

Though different, we still had a blast at last year’s ASMS and we were honored that so many of you chose to join our virtual events. Revisit our virtual booth here