A Cost-Effective High-Throughput Plasma and Serum Proteomics Workflow Enables Mapping of the Molecular Impact of Total Pancreatectomy with Islet Autotransplantation

Bennike, Tue Bjerg; Bellin, Melena D.; Xuan, Yue; Stensballe, Allan; Møller, Frederik Trier; Beilman, Gregory J.; Levy, Ofer; Cruz‐Monserrate, Zobeida; Andersen, Vibeke; Steen, Judith A.; Conwell, Darwin L.; Steen, Hanno

doi:10.1021/acs.jproteome.8b00111

Cited by 39 publications

(66 citation statements)

References 47 publications

(108 reference statements)

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“…We analyzed plasma samples using our published plasma proteomics workflow in a 96-well plate format, and identified 382 plasma proteins (FDR<1%), of which 197 passed our criteria for quantifiable proteins ( 10 , 11 ). These numbers are comparable to those reported in similar plasma- and serum studies ( 10 , 35 , 36 ).…”

Section: Resultsmentioning

confidence: 99%

“…For the original study ( 7 ), plasma samples from newborns were prepared for proteome analysis using the in-house developed plasma and serum proteomics workflow, based on the MStern blotting sample processing and trypsinization protocol ( 10 , 11 ). To this end, 5 µL plasma were diluted in 100 µL sample buffer (8 M urea in TRIS-HCl, pH 8.5).…”

Section: Methodsmentioning

confidence: 99%

“…The relative inter-abundances of the IgG subclasses were estimated using the intensity-based absolute quantitation (iBAQ) values from MaxQuant, calculated from unique peptide abundances only (uploaded to the PRIDE repository PXD019817). To enable a comparison to the adult state, we used data from a previously published proteomics study of plasma from 30 healthy adult Danes ( 10 ). To compensate for the different experimental designs, we normalized the IgG ratios to IgG2 (of note, normalization to IgG3 yielded similar results) in each dataset.…”

Section: Methodsmentioning

confidence: 99%

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Preparing for Life: Plasma Proteome Changes and Immune System Development During the First Week of Human Life

et al. 2020

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Neonates have heightened susceptibility to infections. The biological mechanisms are incompletely understood but thought to be related to age-specific adaptations in immunity due to resource constraints during immune system development and growth. We present here an extended analysis of our proteomics study of peripheral blood-plasma from a study of healthy full-term newborns delivered vaginally, collected at the day of birth and on day of life (DOL) 1, 3, or 7, to cover the first week of life. The plasma proteome was characterized by LC-MS using our established 96-well plate format plasma proteomics platform. We found increasing acute phase proteins and a reduction of respective inhibitors on DOL1. Focusing on the complement system, we found increased plasma concentrations of all major components of the classical complement pathway and the membrane attack complex (MAC) from birth onward, except C7 which seems to have near adult levels at birth. In contrast, components of the lectin and alternative complement pathways mainly decreased. A comparison to whole blood messenger RNA (mRNA) levels enabled characterization of mRNA and protein levels in parallel, and for 23 of the 30 monitored complement proteins, the whole blood transcript information by itself was not reflective of the plasma protein levels or dynamics during the first week of life. Analysis of immunoglobulin (Ig) mRNA and protein levels revealed that IgM levels and synthesis increased, while the plasma concentrations of maternally transferred IgG1-4 decreased in accordance with their in vivo half-lives. The neonatal plasma ratio of IgG1 to IgG2-4 was increased compared to adult values, demonstrating a highly efficient IgG1 transplacental transfer process. Partial compensation for maternal IgG degradation was achieved by endogenous synthesis of the IgG1 subtype which increased with DOL. The findings were validated in a geographically distinct cohort, demonstrating a consistent developmental trajectory of the newborn’s immune system over the first week of human life across continents. Our findings indicate that the classical complement pathway is central for newborn immunity and our approach to characterize the plasma proteome in parallel with the transcriptome will provide crucial insight in immune ontogeny and inform new approaches to prevent and treat diseases.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Preparing for Life: Plasma Proteome Changes and Immune System Development During the First Week of Human Life

et al. 2020

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“…He outlined the objective to develop a plasma proteomics platform that is applicable to small samples, provides high throughput, and is cost efficient. He described a mass spectrometry-based workflow wherein as little as 0.3 µl of plasma or serum enables analysis to a robust analytical depth (53). He presented studies employing these methods to study small sample volumes in the context of immune ontogeny across the first week of life, Lyme Disease biomarker discovery as well as systems vaccinology to characterize responses to hepatitis B and influenza vaccines.…”

Section: Systems Biologymentioning

confidence: 99%

Towards Precision Vaccines: Lessons From the Second International Precision Vaccines Conference

et al. 2020

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Other than clean drinking water, vaccines have been the most effective public health intervention in human history, yet their full potential is still untapped. To date, vaccine development has been largely limited to empirical approaches focused on infectious diseases and has targeted entire populations, potentially disregarding distinct immunity in vulnerable populations such as infants, elders, and the immunocompromised. Over the past few decades innovations in genetic engineering, adjuvant discovery, formulation science, and systems biology have fueled rapid advances in vaccine research poised to consider demographic factors (e.g., age, sex, genetics, and epigenetics) in vaccine discovery and development. Current efforts are focused on leveraging novel approaches to vaccine discovery and development to optimize vaccinal antigen and, as needed, adjuvant systems to enhance vaccine immunogenicity while maintaining safety. These approaches are ushering in an era of precision vaccinology aimed at tailoring immunization for vulnerable populations with distinct immunity. To foster collaboration among leading vaccinologists, government, policy makers, industry partners, and funders from around the world, the Precision Vaccines Program at Boston Children's Hospital hosted the 2 nd International Precision Vaccines Conference (IPVC) at Harvard Medical School on the 17 th-18 th October 2019. The conference convened experts in vaccinology, including vaccine formulation and adjuvantation, immunology, cell signaling, systems biology, biostatistics, bioinformatics, as well as vaccines for non-infectious indications such as cancer and opioid use disorder. Herein we review highlights from the 2 nd IPVC and discuss key concepts in the field of precision vaccines.

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“…Automatization and 96-well plate based sample processing allow the preparation of hundreds of samples per day and reduce batch effects, that otherwise are a challenge in large-scale and longitudinal experiments [6][7][8][9][10][11][12] . Further, fast, efficient and robust chromatographic separations have been achieved by replacing nanoflow LC, as traditionally used in proteomics 13,14 , with setups that use higher flow-rates.…”

Section: Introductionmentioning

confidence: 99%

Scanning SWATH acquisition enables high-throughput proteomics with chromatographic gradients as fast as 30 seconds

Messner

Demichev

Bloomfield

et al. 2019

Preprint

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Rapidly emerging applications in data-driven biology and personalised medicine call for the development of fast and reliable proteomic methods. However, the use of fast chromatographic gradients is limited by the mass spectrometers' sampling rate and signal interferences. Here we present scanningSWATH, a data-independent acquisition method, in which the DIA-typical stepwise windowed acquisition is replaced by continuous scanning with the first quadrupole.ScanningSWATH enables ultra-fast duty cycles as well as to assign precursor masses to the MS2 fragment traces. Furthermore, we have implemented the support for scanningSWATH in DIA-NN, a fully-automated software suite designed to deconvolute fast DIA experiments, which corrects for signal interferences. We show that the combination of scanningSWATH and DIA-NN enables the efficient application of high-flow liquid chromatography (800 µL/min flow rate) to proteomics. High-flow scanningSWATH increases proteomic sample throughput to the minute-scale, while the use of high-flow chromatography hardware improves reliability and robustness. Benchmarking on yeast and human cell lysates, we demonstrate that the proteomic depth achieved with five-minute high-flow scanningSWATH gradients is comparable to that obtained with several times slower nano-and microflow chromatographic gradients, even if compared to most recent studies that used microflow-SWATH or Evosep-DIA methods. The combination of scanningSWATH, advanced data processing, and industry-standard high-flow LC hardware paves a way for a new generation of cheaper and highly consistent proteomic methods. These allow the recording of hundreds of precise quantitative proteomes per day on a single instrument.

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A Cost-Effective High-Throughput Plasma and Serum Proteomics Workflow Enables Mapping of the Molecular Impact of Total Pancreatectomy with Islet Autotransplantation

Cited by 39 publications

References 47 publications

Preparing for Life: Plasma Proteome Changes and Immune System Development During the First Week of Human Life

Preparing for Life: Plasma Proteome Changes and Immune System Development During the First Week of Human Life

Towards Precision Vaccines: Lessons From the Second International Precision Vaccines Conference

Scanning SWATH acquisition enables high-throughput proteomics with chromatographic gradients as fast as 30 seconds

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