FLASHQuant: a fast algorithm for proteoform quantification in top-down proteomics DOI Creative Commons
Jihyung Kim, Kyowon Jeong,

Philipp T. Kaulich

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown

Опубликована: Ноя. 11, 2023

ABSTRACT Liquid chromatography-mass spectrometry (LC-MS) based top-down proteomics (TDP) is an essential method for the analysis of intact proteoforms. The accurate quantification individual proteoforms a crucial step in identifying proteome-wide alterations different biological conditions. Label-free (LFQ) most common proteoform as it requires no additional costly labeling. In TDP, due to frequent co-elution and complex signal structures, overlapping signals deriving from multiple complicate quantification. Here, we introduce FLASHQuant MS1-level LFQ which capable automatically resolving quantifying co-eluting performs highly reproducible short runtimes just few minutes per LC-MS run. To validate reported by FLASHQuant, evaluated them with identified confirmed tandem mass spectrometry, showed high match rates. publicly available platform-independent open-source software at https://openms.org/flashquant/ .

Язык: Английский

Cysteine-Directed Isobaric Labeling Combined with GeLC-FAIMS-MS for Quantitative Top-Down Proteomics DOI Creative Commons

Theo Matzanke,

Philipp T. Kaulich,

Kyowon Jeong

и другие.

Journal of Proteome Research, Год журнала: 2025, Номер unknown

Опубликована: Янв. 30, 2025

The quantification of proteoforms, i.e., all molecular forms in which proteins can be present, by top-down proteomics provides essential insights into biological processes at the level. Isobaric labeling-based strategies are suitable for multidimensional separation and allow multiplexing samples. Here, we investigated cysteine-directed isobaric labeling iodoTMT combination with a gel- gas-phase fractionation (GeLC-FAIMS-MS) in-depth quantitative proteoform analysis. We optimized acquisition workflow (i.e., FAIMS compensation voltages, isolation windows, strategy, fragmentation method) using two-proteome mix to increase number quantified proteoforms reduce ratio compression. Additionally, implemented mass feature-based strategy widely used deconvolution algorithm FLASHDeconv, improves facilitates data GeLC-FAIMS-MS was applied quantitatively analyze proteome Escherichia coli grown under glucose or acetate as sole carbon source, resulting identification 726 differentially abundant proteoforms.

Язык: Английский

Процитировано

1
Diversifying the concept of model organisms in the age of -omics DOI Creative Commons
Fabrice Bertile, Sabine Matallana‐Surget, Andreas Tholey

и другие.

Communications Biology, Год журнала: 2023, Номер 6(1)

Опубликована: Окт. 19, 2023

Abstract In today’s post-genomic era, it is crucial to rethink the concept of model organisms. While a few historically well-established organisms, e.g. laboratory rodents, have enabled significant scientific breakthroughs, there now pressing need for broader inclusion. Indeed, new organisms and models, from complex microbial communities holobionts, are essential fully grasp complexity biological principles across breadth biodiversity. By fostering collaboration between biology, advanced molecular science omics communities, we can collectively adopt unraveling their functioning, uncovering fundamental mechanisms. This concerted effort will undoubtedly enhance human health, environmental quality, biodiversity conservation.

Язык: Английский

Процитировано

17
Orbitrap Mass Spectrometry and High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Enable the in-Depth Analysis of Human Serum Proteoforms DOI Creative Commons
Jake Kline,

Michael W. Belford,

Cornelia L. Boeser

и другие.

Journal of Proteome Research, Год журнала: 2023, Номер 22(11), С. 3418 - 3426

Опубликована: Сен. 29, 2023

Blood serum and plasma are arguably the most commonly analyzed clinical samples, with dozens of proteins serving as validated biomarkers for various human diseases. Top-down proteomics may provide additional insights into disease etiopathogenesis since this approach focuses on protein forms, or proteoforms, originally circulating in blood, potentially providing access to information about relevant post-translational modifications, truncations, single amino acid substitutions, many other sources variation. However, vast majority proteomic studies carried out using peptide-centric, bottom-up approaches that cannot recapitulate original proteoform content samples. Clinical laboratories have been slow adopt top-down analysis, also due higher sample handling requirements. In study, we describe a straightforward protocol intact preparation based depletion albumin immunoglobulins, followed by simplified fractionation via polyacrylamide gel electrophoresis. After molecular weight-based fractionation, supplemented traditional liquid chromatography–tandem mass spectrometry (LC-MS2) data acquisition high-field asymmetric waveform ion mobility (FAIMS) further simplify mixtures. This LC-FAIMS-MS2 method led identification over 1000 proteoforms < 30 kDa, outperforming LC-MS2 more than doubling number identified previous studies.

Язык: Английский

Процитировано

13
Droplet-Based Microfluidics with Mass Spectrometry for Microproteomics DOI Creative Commons
Hang Li,

Yudan Ma,

Rongxin Fu

и другие.

Engineering, Год журнала: 2024, Номер unknown

Опубликована: Сен. 1, 2024

Язык: Английский

Процитировано

4
Dynamic whole genome amplification using digital microfluidics for single-cell sequencing DOI
Qingyu Ruan,

Changrui Shi,

Xiaoye Lin

и другие.

Chemical Engineering Journal, Год журнала: 2025, Номер unknown, С. 162099 - 162099

Опубликована: Март 1, 2025

Язык: Английский

Процитировано

0
Top‐Down Proteomics: Why and When? DOI Creative Commons

Philipp T. Kaulich,

Andreas Tholey

PROTEOMICS, Год журнала: 2025, Номер unknown

Опубликована: Апрель 27, 2025

ABSTRACT Manifold biological processes at all levels of transcription and translation can lead to the formation a high number different protein species (i.e., proteoforms), which outnumber sequences encoded in genome by far. Due large molecules formed this way, span an enormous range physicochemical properties, proteoforms are functional drivers processes, creating need for powerful analytical approaches decipher language life. While bottom‐up proteomics has become most widely used approach, providing features such as sensitivity, depth analysis, throughput, it its limitations when comes identifying, quantifying, characterizing proteoforms. In particular, major bottleneck is assign peptide‐level information original contrast, top‐down (TDP) targets direct analysis intact Despite being characterized technological challenges, TDP community established numerous protocols that allow easy implementation any laboratory. viewpoint, we compare both approaches, argue worth embedding experiments, show fields research be successfully implemented perform integrative multi‐level proteoformics.

Язык: Английский

Процитировано

0
Boronic Acid‐Rich Lanthanide Metal‐Organic Frameworks Enable Deep Proteomics with Ultratrace Biological Samples DOI
Shuang Zhang, Behafarid Ghalandari,

Youming Chen

и другие.

Advanced Materials, Год журнала: 2024, Номер 36(33)

Опубликована: Июль 3, 2024

Label-free proteomics is widely used to identify disease mechanism and potential therapeutic targets. However, deep with ultratrace clinical specimen remains a major technical challenge due extensive contact loss during complex sample pretreatment. Here, hybrid of four boronic acid-rich lanthanide metal-organic frameworks (MOFs) high protein affinity introduced capture proteins in samples jointly by nitrogen-boronate complexation, cation-π ionic interactions. A MOFs Aided Sample Preparation (MASP) workflow that shrinks volume integrates lysis, capture, digestion peptide collection steps into single PCR tube minimize caused non-specific absorption, proposed further. MASP validated quantify ≈1800 10 HEK-293T cells. applied profile cerebrospinal fluid (CSF) proteome from cerebral stroke brain damaged patients, identified ≈3700 1 µL CSF. further demonstrated detect ≈9600 as few 50 µg mouse tissues. thus enables deep, scalable, reproducible on precious low abundant proteins.

Язык: Английский

Процитировано

3
Digital Microfluidics with Integrated Raman Sensor for High-Sensitivity In-Situ Bioanalysis DOI
Wenbo Dong, Rongxin Fu, Nan Zhang

и другие.

Biosensors and Bioelectronics, Год журнала: 2024, Номер 271, С. 117036 - 117036

Опубликована: Дек. 4, 2024

Язык: Английский

Процитировано

3
Superparamagnetic Composite Nanobeads Anchored with Molecular Glues for Ultrasensitive Label‐free Proteomics DOI
Shuang Zhang, Behafarid Ghalandari, Aiting Wang

и другие.

Angewandte Chemie International Edition, Год журнала: 2023, Номер 62(45)

Опубликована: Сен. 1, 2023

Abstract Mass spectrometry has emerged as a mainstream technique for label‐free proteomics. However, proteomic coverage trace samples is constrained by adsorption loss during repeated elution at sample pretreatment. Here, we demonstrated superparamagnetic composite nanoparticles functionalized with molecular glues (MGs) to enrich proteins in human biofluid. We showed high protein binding (>95 %) and recovery (≈90 rates anchor‐nanoparticles. further proposed Streamlined Workflow based on Anchor‐nanoparticles Proteomics (SWAP) method that enabled unbiased capture, digestion pure peptides one single tube. SWAP quantify over 2500 groups 100 HEK 293T cells. adopted profile proteomics aqueous humor from cataract ( n =15) wet age‐related macular degeneration =8) patients, quantified ≈1400 5 μL humor. simplifies preparation steps, minimizes improves previous samples.

Язык: Английский

Процитировано

8
Lectin-Based SP3 Technology Enables N-Glycoproteomic Analysis of Mouse Oocytes DOI

Zian Huo,

Haixia Tu,

Jie Ren

и другие.

Journal of Proteome Research, Год журнала: 2024, Номер 23(6), С. 2137 - 2147

Опубликована: Май 24, 2024

N-glycosylation is one of the most universal and complex protein post-translational modifications (PTMs), it involved in many physiological pathological activities. Owing to low abundance N-glycoproteins, enrichment N-glycopeptides for mass spectrometry analysis usually requires a large amount peptides. Additionally, oocyte has not been systemically characterized due limited sample amount. Here, we developed glycosylation method based on lectin single-pot, solid-phase-enhanced preparation (SP3) technology, termed lectin-based SP3 technology (LectinSP3). LectinSP3 immobilized beads N-glycopeptide enrichment. It could identify over 1100 sites 600 N-glycoproteins from 10 μg mouse testis Furthermore, using method, N-glycoproteome 1000 oocytes three replicates identified total 363 215 N-glycoproteins. Bioinformatics revealed that these were mainly enriched cell adhesion, fertilization, sperm-egg recognition. Overall, all procedures performed tube, magnetic beads. suitable samples expected be easily adaptable automation. In addition, our profiling help further characterize regulation functions.

Язык: Английский

Процитировано

2