Dataset Information

Rapid tryptic peptide mapping of human serum albumin using DI-MS/MS^ALL.

ABSTRACT: In recent decades, proteinic drugs, in particular monoclonal antibodies, are taking the leading role of small molecule drugs, and peptide mapping relying on liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an emerging approach to substitute the role of a ligand-binding assay for the quality control of the proteinic drugs. However, such LC-MS/MS approaches extensively suffer from time-intensive measurements, leading to a limited throughput. To achieve accelerated measurements, here, the potential of DI-MS/MS^ALL towards tryptic peptide mapping was evaluated through comparing with well-defined LC-MS/MS means, and human serum albumin (HSA) was employed as the representative protein for applicability illustration. Among the 55 tryptic peptides theoretically suggested by Skyline software, 47 were successfully captured by DI-MS/MS^ALL through acquiring the desired MS² spectra, in comparison to 51 detected by LC-MS/MS. DI-MS/MS^ALL measurements merely took 5 min, which was dramatically superior to the LC-MS/MS assay. Noteworthily, different from fruitful multi-charged MS¹ signals for LC-MS/MS, most quasi-molecular ions received lower charged states. DI-MS/MS^ALL also possessed advantages such as lower solvent consumption and facile instrumentation; however, more sample was consumed. In conclusion, DI-MS/MS^ALL is eligible to act as an alternative analytical tool for LC-MS/MS towards the peptide mapping of proteinic drugs, particularly when a heavy measurement workload.

SUBMITTER: Zhang K

PROVIDER: S-EPMC8963265 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Rapid tryptic peptide mapping of human serum albumin using DI-MS/MS<sup>ALL</sup>.

Zhang Ke K Gong Xingcheng X Wang Qian Q Tu Pengfei P Li Jun J Song Yuelin Y

RSC advances 20220329 16

In recent decades, proteinic drugs, in particular monoclonal antibodies, are taking the leading role of small molecule drugs, and peptide mapping relying on liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an emerging approach to substitute the role of a ligand-binding assay for the quality control of the proteinic drugs. However, such LC-MS/MS approaches extensively suffer from time-intensive measurements, leading to a limited throughput. To achieve accelerated measurements, here, t ...[more]

PMID: 35424948

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Project description:The biological significance of chemical modifications to the ribonucleic acid (RNA) of human immunodeficiency virus type-1 (HIV-1) has been recognized. However, our understanding of the site-specific and context-dependent roles of these chemical modifications remains limited, primarily due to the absence of nucleotide-resolution mapping of modification sites. In this study, we present a method for achieving nucleotide-resolution mapping of chemical modification sites on HIV-1 RNA using liquid chromatography and tandem mass spectrometry (LC-MS/MS). LC-MS/MS, a powerful tool capable of directly analyzing native RNAs, has proven effective for mapping RNA modifications in small RNA molecules, including ribosomal RNA and transfer RNA. However, longer RNAs have posed challenges, such as the 9 Kb HIV-1 virion RNA, due to the complexity of and ambiguity in mass differences among RNase T1-cleaved RNA fragments in LC-MS/MS data. Here, we introduce a new target RNA enrichment method to isolate small local RNA fragments of HIV-1 RNA that potentially harbor site-specific N6-methyladenosine (m6A) modifications. In our initial trial, we used target-specific DNA probes only and encountered insufficient RNA fragmentation due to inefficient S1 digestion near the target site. Recognizing that inefficient S1 digestion by HIV-1 RNA is likely due to the formation of secondary structures in proximity to the target site, we designed multiple DNA probes annealing to various sites of HIV-1 RNA to better control the structures of RNA substrates for S1 digestion. The use of these non-target DNA probes significantly improved the isolation of more homogeneous target RNA fragments of approximately 50 bases in length. Oligonucleotide LC-MS/MS analysis of these isolated target RNA fragments successfully separated and detected both m6A-methylated and non-methylated oligomers at the two m6A-predicted sites. The principle of this new target enrichment strategy holds promise and should be broadly applicable to the analysis of any lengthy RNA that was previously deemed infeasible for investigation using oligonucleotide LC-MS/MS.

Dataset Information

Rapid tryptic peptide mapping of human serum albumin using DI-MS/MS^ALL.

Publications

Rapid tryptic peptide mapping of human serum albumin using DI-MS/MS<sup>ALL</sup>.

Similar Datasets

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Dataset Information

Rapid tryptic peptide mapping of human serum albumin using DI-MS/MSALL.

Publications

Rapid tryptic peptide mapping of human serum albumin using DI-MS/MS<sup>ALL</sup>.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Rapid tryptic peptide mapping of human serum albumin using DI-MS/MS^ALL.