Targeted liquid chromatography‐tandem mass spectrometry analysis of proteins: Basic principles, applications, and perspectives

Kulyyassov, Arman; Fresnais, Margaux; Longuespée, Rémi

doi:10.1002/pmic.202100153

Cited by 25 publications

(20 citation statements)

References 175 publications

(199 reference statements)

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“…Sox2 has HMG domain, and Oct4 has two POU HD and POU S domains that help these transcription factors to assemble on composite DNA motifs of regulatory elements of genes such as Utf1 or FGF4 [44]. Due to this DNA-dependent binding and interaction of these proteins on such motifs, this results in the labeling of the BAP target and high level of biotinylation detected by MRM mode [45] of LC-MS/MS and streptavidin Western blotting in comparison with controls, where we used GFP or Tap54beta fusions of BAP (Figure 3D). Quantification of MRM data was performed using Skyline software [46].…”

Section: Examples Of Applications Of Pub Methodsmentioning

confidence: 99%

Generation of Peptides for Highly Efficient Proximity Utilizing Site-Specific Biotinylation in Cells

Kulyyassov

Ramankulov

Ogryzko

2022

Life

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Protein tags are peptide sequences genetically embedded into a recombinant protein for various purposes, such as affinity purification, Western blotting, and immunofluorescence. Another recent application of peptide tags is in vivo labeling and analysis of protein–protein interactions (PPI) by proteomics methods. One of the common workflows involves site-specific in vivo biotinylation of an AviTag-fused protein in the presence of the biotin ligase BirA. However, due to the rapid kinetics of labeling, this tag is not ideal for analysis of PPI. Here we describe the rationale, design, and protocol for the new biotin acceptor peptides BAP1070 and BAP1108 using modular assembling of biotin acceptor fragments, DNA sequencing, transient expression of proteins in cells, and Western blotting methods. These tags were used in the Proximity Utilizing Biotinylation (PUB) method, which is based on coexpression of BAP-X and BirA-Y in mammalian cells, where X or Y are candidate interacting proteins of interest. By changing the sequence of these peptides, a low level of background biotinylation is achieved, which occurs due to random collisions of proteins in cells. Over 100 plasmid constructs, containing genes of transcription factors, histones, gene repressors, and other nuclear proteins were obtained during implementation of projects related to this method.

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Section: Examples Of Applications Of Pub Methodsmentioning

confidence: 99%

Generation of Peptides for Highly Efficient Proximity Utilizing Site-Specific Biotinylation in Cells

Kulyyassov

Ramankulov

Ogryzko

2022

Life

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“…Other examples are information resources containing a large amount of data obtained in the course of experiments on targeted proteomics [20]. These databases can help select suitable candidate peptides for the development of peptide quantitation methods using the Multiple reaction monitoring (MRM) method.…”

Section: Specialized Databasesmentioning

confidence: 99%

Uniprot Database - Universal Information Resource of Protein Sequences

Kulyyassov¹

2022

Eurasian Journal of Applied Biotechnology

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Protein sequences are stored in public databases such as the UniProt Knowledgebase (UniProtKB), where curators add bioinformatics data, including prediction of structure and function of biomolecules and experimental results. Protein function prediction can be done using sequence similarity searches, but an alternative approach is to use protein signatures that classify proteins into families and domains. The main protein signature databases are accessible through the integrated InterPro database, which provides the UniProtKB sequence classification. In addition to characterizing proteins through protein families, many researchers are interested in analyzing the complete set of proteins from the genome (i.e., the proteome), and there are databases and resources providing unreduced sets of proteomes and analyzes of proteins from organisms with fully sequenced genomes. This article reviews the tools and resources available on the Internet for characterizing both individual proteins and analysis of the entire proteome.

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“…[1][2][3][4][5][6] In addition, by utilizing a multiple reaction monitoring (MRM) scan mode, an LC-MS/MS method is considered the gold standard for quantification analyses of known compounds. [7][8][9] To obtain precise quantified results from MRM chromatograms, it is important that repeatability in an LC-MS/MS system is suitably assessed.…”

Section: Introductionmentioning

confidence: 99%

“…A liquid chromatography system hyphenated with tandem mass spectrometry (LC–MS/MS) system is widely used for the quantification analyses of chemical substances and biological components in various real samples 1–6 . In addition, by utilizing a multiple reaction monitoring (MRM) scan mode, an LC–MS/MS method is considered the gold standard for quantification analyses of known compounds 7–9 . To obtain precise quantified results from MRM chromatograms, it is important that repeatability in an LC–MS/MS system is suitably assessed.…”

Section: Introductionmentioning

confidence: 99%

Effects of digital processing on repeatability assessment of a multiple reaction monitoring liquid chromatography–tandem mass spectrometry system by ISO 11843‐7

Machida

Kotani

Hayashi³

et al. 2022

J Mass Spectrom

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ISO 11843 part 7 (ISO 11843-7) can provide a standard deviation (SD) of area measurements of a target peak through the stochastic behaviors of instrumental noises. The purpose of this study is to demonstrate that ISO 11843-7 can be applied to assess repeatability in an isocratic liquid chromatography-tandem mass spectrometry (LC-MS/MS) system without repetitive measurements. The relative standard deviation (RSD) of the peak area of ergosterol picolinyl ester, which was used as an example, on a multiple reaction monitoring (MRM) chromatogram was determined by ISO 11843-7. The RSD by ISO 11843-7 (N = 1) was within a 95% confidence band of the RSD by repetitive measurements (N = 6). Moreover, the effects of digital smoothing, such as moving average, were also examined on the repeatability assessment in LC-MS/MS by ISO 11843-7. From the results of the comparisons of the RSDs obtained by ISO 11843-7 and the repetitive measurements, it was shown that suitable RSDs of the peak area were obtained from the smoothed MRM chromatograms by the moving average for narrow data point windows (e.g., one-sixth of the peak width). In conclusion, the utility of repeatability assessment based on ISO 11843-7 has been expanded for the validation of an LC-MS/MS system.

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Targeted liquid chromatography‐tandem mass spectrometry analysis of proteins: Basic principles, applications, and perspectives

Cited by 25 publications

References 175 publications

Generation of Peptides for Highly Efficient Proximity Utilizing Site-Specific Biotinylation in Cells

Generation of Peptides for Highly Efficient Proximity Utilizing Site-Specific Biotinylation in Cells

Uniprot Database - Universal Information Resource of Protein Sequences

Effects of digital processing on repeatability assessment of a multiple reaction monitoring liquid chromatography–tandem mass spectrometry system by ISO 11843‐7

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