Application of Displacement Chromatography to Online Two-Dimensional Liquid Chromatography Coupled to Tandem Mass Spectrometry Improves Peptide Separation Efficiency and Detectability for the Analysis of Complex Proteomes

Kwiatkowski, Marcel; Krösser, Dennis; Wurlitzer, M.; Steffen, Pascal; Barcaru, Andrei; Horvatovich, Péter; Bischoff, Rainer; Schlüter, Hartmut

doi:10.1021/acs.analchem.8b02189

Cited by 18 publications

(12 citation statements)

References 28 publications

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“…In the present 2D-LC system, an online automatic wash controller was employed, which was not reported in the previous studies of the 2D-LC system (Bob et al, 2019;Kwiatkowski et al, 2018). To avoiding the interference of impurities, the automatic wash system accomplished cleaning of the columns and the total pipeline online after each injection.…”

Section: Resultsmentioning

confidence: 99%

A novel and sensitive method for determination of amisulpride in human plasma by two‐dimensional liquid chromatography

Wang

et al. 2021

Biomedical Chromatography

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A novel and sensitive heart-cutting two-dimensional liquid chromatography with ultraviolet detection method (2D-LC-UV) was developed and validated for determination of amisulpride in human plasma. The 2D-LC system consists of a first dimensional ( 1 D) LC column and a middle transfer column as well as a second-dimensional ( 2 D) LC column. After simple protein precipitation, the sample was directly injected into the introduction valve of the 2D-LC system. The 1 D column, playing a role of primary separation and preconcentration for complex plasma matrices, transferred the targets to the intermediate column. Following capture of targets on the middle column online, the analytes were transferred to the 2 D separation column by a six-port valve. The 2 D column, avoiding interference from the plasma matrix, completed further separation and quantification. An assistant pump was optimized for primary enrichment as well as final elution in the heart-cutting mode. The analytical time of amisulpride was 7.401 min. The accuracy was between 0.48 and 8.49%, while the intra-and inter-day precisions ranged from 0.9 to 3.1% and from 1.7% to 3.3%, respectively. The linear range of amisulpride was 48.15-2,407.59 ng/ml, while the extraction recovery was 98.7-101.3%. The strategy established in the study, which was successfully applied to therapeutic drug monitoring of amisulpride for routine clinical detection, displays high sensitivity, good repeatability, convenience and low cost.

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Section: Resultsmentioning

confidence: 99%

A novel and sensitive method for determination of amisulpride in human plasma by two‐dimensional liquid chromatography

Wang

et al. 2021

Biomedical Chromatography

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“…Following vacuum‐centrifugation and subsequent reconstitution in 0.1% formic acid (FA), peptides were separated by RPLC (C18; buffer A: 0.1% FA dissolved in HPLC‐H 2 O; buffer B: 0.1% FA, dissolved in ACN) using a flow rate of 0.4 µL/min and a gradient of 2–30% in 30 min. Eluting peptides were introduced via an ESI interface into a Q‐TOF mass spectrometer (one boar, Q‐TOF Ultima, Micromass/Waters, Manchester, UK) and an ion‐trap mass spectrometer (two other boars, XCT ion‐trap, Agilent Technologies, Waldbronn, Germany), and analyzed as described elsewhere [49, 50].…”

Section: Methodsmentioning

confidence: 99%

Protein speciation is likely to increase the chance of proteins to be determined in 2‐DE/MS

et al. 2022

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Multiple spotting due to protein speciation might increase a protein's chance of being captured in a random selection of 2-DE spots. We tested this expectation in new (PXD015649) and previously published 2-DE/MS data of porcine and human tissues. For comparison, we included bottom-up proteomics studies (BU-LC/MS) of corresponding biological materials. Analyses of altogether ten datasets proposed that amino acid modification fosters multispotting in 2-DE. Thus, the number of 2-DE spots containing a particular protein more tightly associated with a peptide diversity measure accounting for amino acid modification than with an alternative one disregarding it. Furthermore, every 11th amino acid was a post-translational modification candidate site in 2-DE/MS proteins, whereas in BU-LC/MS proteins this was merely the case in every 21st amino acid. Alternative splicing might contribute to multispotting, since genes encoding 2-DE/MS proteins were found to have on average about 0.3 more transcript variants than their counterparts from BU-LC/MS studies. Correspondingly, resolution completeness as estimated from the representation of transcript variant-rich genes was higher in 2-DE/MS than BU-LC/MS datasets. These findings suggest that the ability to resolve proteomes down to protein species can lead to enrichment of multispotting proteins in 2-DE/MS. Low sensitivity of stains and MS instruments appears to enhance this effect. K E Y W O R D Salternative splicing, post-translational modification, protein abundance, protein species, twodimensional gel electrophoresis Abbreviations: AS, Alternative splicing; 2-DE/MS, 2-DE followed by mass spectrometry; BU-LC/MS, bottom-up proteomics based on liquid chromatography coupled to MS; IEF, isoelectric focussing; ∆PD, extent to which post-translational modifications increase peptide diversity; FA, formic acid; FDR, False discovery rate; GO, gene ontology; N TV , number of transcript variants per gene; PD, peptide diversity with modifications disregarded; PMI, post-translational modification index; PTM, post-translational modification; RT, room temperature; TPM, transcripts per million.

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“…Within a band a high purity of the component is achieved [72]. DE has been shown to be suitable for separation of complex mixture of tryptic peptides [73][74][75] and proteins [76][77][78][79]. One of the characteristics of DE is that DE has a different selectivity compared with GE [77].…”

Section: Separation Of Proteoforms Of Therapeutic Proteins 41 Separamentioning

confidence: 99%

Preparing Proteoforms of Therapeutic Proteins for Top-Down Mass Spectrometry

Hidayah¹,

Gaikwad²,

Heikaus³

et al. 2020

Proteoforms - Concept and Applications in Medical Sciences

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A characteristic of many proteoforms, derived from a single gene, is their similarity regarding the composition of atoms, making their analysis very challenging. Many overexpressed recombinant proteins are strongly associated with this problem, especially recombinant therapeutic glycoproteins from large-scale productions. In contrast to small molecule drugs, which consist of a single defined molecule, therapeutic protein preparations are heterogenous mixtures of dozens or even hundreds of very similar species. With mass spectrometry, currently highquality spectra of intact proteoforms can be obtained only, if the complexity of the mixture of individual proteoform-ions, entering the gas phase at the same time is low. Thus, prior to mass spectrometric analysis, an effective separation is required for getting fractions with a low number of individual proteoforms. This is especially true not only for recombinant therapeutic proteins, because of their huge heterogeneity, but also relevant for top-down proteomics. Purification of proteoforms is the bottleneck in analyzing intact proteoforms with mass spectrometry. This review is focusing on the current state of the art, especially of liquid chromatography for preparing proteoforms for mass spectrometric top-down analysis. The topic of therapeutic proteins has been chosen, because this group of proteins is most challenging regarding their proteoform analysis.

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Application of Displacement Chromatography to Online Two-Dimensional Liquid Chromatography Coupled to Tandem Mass Spectrometry Improves Peptide Separation Efficiency and Detectability for the Analysis of Complex Proteomes

Cited by 18 publications

References 28 publications

A novel and sensitive method for determination of amisulpride in human plasma by two‐dimensional liquid chromatography

A novel and sensitive method for determination of amisulpride in human plasma by two‐dimensional liquid chromatography

Protein speciation is likely to increase the chance of proteins to be determined in 2‐DE/MS

Preparing Proteoforms of Therapeutic Proteins for Top-Down Mass Spectrometry

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