Journal of Proteome Research, Год журнала: 2024, Номер unknown
Опубликована: Ноя. 12, 2024
Tandem mass spectrometry (MS/MS) is the gold standard for intact glycopeptide identification, enabling peptide sequence elucidation and site-specific localization of glycan compositions. Beam-type collisional activation generally sufficient
Язык: Английский
Nature Communications, Год журнала: 2025, Номер 16(1)
Опубликована: Март 13, 2025
Abstract Recently, a conceptually new mass analyzer was introduced by pairing quadrupole Orbitrap spectrometer with an asymmetric track lossless (Astral™) analyzer. This system provides >200 Hz MS/MS scanning speed, high resolving power, sensitivity, and accuracy. Due to its the instrument allows for narrow-window data-independent acquisition (nDIA) strategy, representing technical milestone in peptide-centric proteomics. However, this may also be applied other complex clinically important proteomes, such as human plasma N -glycoproteome. Here, we evaluate Astral in-depth analysis of -glycoproteome pioneer dedicated nDIA workflow, termed “nGlycoDIA”, on glycopeptide enriched crude plasma. strategy leads cumulative identification over 3000 unique glycoPSMs derived from 181 glycoproteins just 40 minutes covers dynamic range 7 orders magnitude sample. Notably, detect several glycosylated cytokines that have reported concentrations ng/L range. Furthermore, shortening gradient 10 min still detection almost 1850 (95% CI [1840-1860]) glycoPSMs, indicating high-throughput clinical glycoproteomics within reach.
Язык: Английский
Процитировано
0
bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown
Опубликована: Апрель 16, 2025
ABSTRACT Current applications of mass spectrometry-based proteomics range from single cell to body fluid analysis that come with very different demands regarding sensitivity or sample throughput. Additionally, the vast molecular complexity proteomes and massive dynamic protein concentrations in these biological systems require high-performance chromatographic separations tandem high speed afforded by spectrometer. In this study, we focussed on angle and, more specifically, systematically evaluated proteome performance across a wide flow rates (0.3 – 50 μL/min) associated column diameters using Vanquish Neo UHPLC coupled online Q Exactive HF-X Serial dilutions HeLa line digests were used for benchmarking total time injection-to-injection was intentionally fixed at 60 minutes (24 samples per day). The three key messages study are i) all suitable high-quality analysis, ii) capLC (1.5 is robust, sensitive quantitative alternative nanoLC many iii) showcased proteome, phosphoproteome drug data provide sound empirical guidance laboratories selecting appropriate their specific application.
Язык: Английский
Процитировано
0
Journal of Chromatography A, Год журнала: 2025, Номер unknown, С. 465976 - 465976
Опубликована: Апрель 1, 2025
Язык: Английский
Процитировано
0
Journal of Proteome Research, Год журнала: 2024, Номер 23(7), С. 2661 - 2673
Опубликована: Июнь 18, 2024
The analysis of the structures glycans present on glycoproteins is an essential component for determining glycoprotein function; however, detailed glycan structural assignment glycopeptides from proteomics mass spectrometric data remains challenging. Glycoproteomic by spectrometry currently can provide significant, yet incomplete, information about present, including monosaccharide composition and in some circumstances site(s) glycosylation. Advancements resolution, using high-mass accuracy instrumentation tailored MS/MS fragmentation parameters, coupled with a dedicated definition diagnostic ions have enabled determination features, or glycotopes, expressed glycopeptides. Here we collation fragments produced traditional positive-ion-mode reversed-phase LC-ESI proteomic workflows describe specific energy settings required to identify glycotopes presented N- O-linked typical experiment.
Язык: Английский
Процитировано
2
Cell, Год журнала: 2024, Номер unknown
Опубликована: Окт. 1, 2024
Язык: Английский
Процитировано
2
Опубликована: Авг. 29, 2024
Hardware changes introduced on the Orbitrap Ascend MultiOmics Tribrid MS include dual ion routing multipoles (IRMs) that can enable parallelized accumulation, dissociation, and mass analysis of three separate populations. The balance between these instrument functions is especially important in glycoproteomics, where complexities glycopeptide fragmentation necessitate large precursor populations and, consequently, long accumulation times for quality MS/MS spectra. To compound matters further, dissociation methods like electron transfer (ETD) benefit characterization come with overhead also slow down scan acquisition. Here we explored how IRM architecture improve analysis, a focus O-glycopeptide using ETD supplemental collisional activation (EThcD). We found parallelization EThcD – uniquely enabled by increased acquisition speed without sacrificing spectral quality, subsequently increasing number O-glycopeptides identified relative to analyses Eclipse (i.e., previous generation MS). Additionally, systematically evaluated ion-ion reaction energies used understand best utilize time architecture. observed shorter-than-expected minimized c/z•-fragment generation, higher collision generate combinations glycan-retaining glycan-neutral-loss peptide backbone fragments identification. saw improvements N-glycopeptide collision-based faster speeds. Overall, data show architectural platform glycoproteomic experiments parallelizing minimize sensitivity.
Язык: Английский
Процитировано
1
bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown
Опубликована: Окт. 15, 2024
Abstract Fragment ion indexing has significantly improved the efficiency of proteomics database search tools. This work implements fragment in Comet, a widely-used, open-source engine. We demonstrate that this enhancement maintains scoring and identification accuracy while substantially increasing peptide spectral matching speeds across multiple applications, including open modification searches, immunopeptidomics, real-time searches. Comet-FI reduced by up to 94% enabling rapid analysis complex data types like immunopeptidomes. Fragment-ion enables Comet keep pace with modern instrumentation expanding applications proteomics, reinforcing its utility diverse workflows integration wide range tools platforms.
Язык: Английский
Процитировано
1
Mass Spectrometry Reviews, Год журнала: 2024, Номер unknown
Опубликована: Сен. 22, 2024
ABSTRACT Nucleic acids are fundamental biological molecules that encode and convey genetic information within living organisms. Over 150 modifications have been found in nucleic acids, which involved critical functions, including regulating gene expression, stabilizing acid structure, modulating protein translation, so on. The dysregulation of is correlated with many diseases such as cancers neurological disorders. However, it still challenging to simultaneously characterize quantify diverse using traditional genomic methods. Mass spectrometry (MS) has served a crucial tool solve this issue, can directly identify the modified species through their distinct mass differences compared canonical ones provide accurate quantitative information. This review surveys history modification discovery, advancements MS‐based methods, sample preparation, applications medical research. We expect high‐throughput valuable from MS analysis will be more broadly applied studying status different pathological conditions, key filling gaps genomics transcriptomics research enabling researchers gain insights into epigenetics epitranscriptomics.
Язык: Английский
Процитировано
0
Опубликована: Авг. 26, 2024
Tandem mass spectrometry (MS/MS) is the gold standard for intact glycopeptide identification, enabling peptide sequence elucidation and site-specific localization of glycan compositions. Beam-type collisional activation generally sufficient N-glycopeptides, while electron-driven dissociation crucial site in O-glycopeptides. Modern glycoproteomic methods often employ multiple techniques within a single LC-MS/MS analysis, but this approach frequently sacrifices sensitivity when analyzing classes simultaneously. Here we explore utility intelligent data acquisition glycoproteomics through real-time library searching (RTLS) to match oxonium ion patterns on-the-fly selection appropriate method. By matching method with class, autonomous dissociation-type (ADS) generates equivalent numbers N-glycopeptide identifications relative traditional beam-type also yielding comparable site-localized O-glycopeptide conventional electron transfer dissociation-based methods. The ADS represents step forward throughput by characterization both N-and O-glycopeptides same acquisition.
Язык: Английский
Процитировано
0
Analytical Chemistry, Год журнала: 2024, Номер 96(37), С. 14867 - 14876
Опубликована: Сен. 6, 2024
Mass spectrometry (MS) using an electron multiplier for intact protein analysis remains limited. Because of the massive size and complex structure proteins, slow flight speed their ions results in few secondary electrons thus low detection sensitivity poor spectral resolution. Thus, we present a compact ion trap-mass approach to directly detect packets obtain high-resolution molecular signature proteins. The disturbances causing deviations motion mass conversion have been clarified advance. radio frequency waveform used manipulate is proposed be sequence constant-frequency steps, interconnected by short time-outs, resulting least dispersive distortion. Furthermore, more such constant-phase conjunctions are arranged each step compensate fluctuations from defects system operation. In addition, two auxiliary pulses generated right phase select specific secular state one clean sharp line.This study demonstrates top-down MS measurement cytochrome C molecules, profile its natural at resolution 20 Da. Additionally, quick scans other proteins were performed.
Язык: Английский
Процитировано
0