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Comprehensive Two-Dimensional Liquid Chromatography-High-Resolution Mass Spectrometry for Complex Protein Digest Analysis Using Parallel Gradients.
van den Hurk, Rick S; Lagerwaard, Bart; Terlouw, Nathan J; Sun, Mingzhe; Tieleman, Job J; Verstegen, Anniek X; Samanipour, Saer; Pirok, Bob W J; Gargano, Andrea F G.
Affiliation
  • van den Hurk RS; Analytical Chemistry Group, Van't Hoff Institute for Molecular Sciences, University of Amsterdam, Amsterdam1098 XH,The Netherlands.
  • Lagerwaard B; Centre for Analytical Sciences Amsterdam (CASA), Amsterdam1098 XH,The Netherlands.
  • Terlouw NJ; Analytical Chemistry Group, Van't Hoff Institute for Molecular Sciences, University of Amsterdam, Amsterdam1098 XH,The Netherlands.
  • Sun M; Centre for Analytical Sciences Amsterdam (CASA), Amsterdam1098 XH,The Netherlands.
  • Tieleman JJ; Analytical Chemistry Group, Van't Hoff Institute for Molecular Sciences, University of Amsterdam, Amsterdam1098 XH,The Netherlands.
  • Verstegen AX; Centre for Analytical Sciences Amsterdam (CASA), Amsterdam1098 XH,The Netherlands.
  • Samanipour S; Analytical Chemistry Group, Van't Hoff Institute for Molecular Sciences, University of Amsterdam, Amsterdam1098 XH,The Netherlands.
  • Pirok BWJ; Centre for Analytical Sciences Amsterdam (CASA), Amsterdam1098 XH,The Netherlands.
  • Gargano AFG; Analytical Chemistry Group, Van't Hoff Institute for Molecular Sciences, University of Amsterdam, Amsterdam1098 XH,The Netherlands.
Anal Chem ; 96(22): 9294-9301, 2024 06 04.
Article de En | MEDLINE | ID: mdl-38758734
ABSTRACT
Despite the high gain in peak capacity, online comprehensive two-dimensional liquid chromatography coupled with high-resolution mass spectrometry (LC × LC-HRMS) has not yet been widely applied to the analysis of complex protein digests. One reason is the method's reduced sensitivity which can be linked to the high flow rates of the second separation dimension (2D). This results in higher dilution factors and the need for flow splitters to couple to ESI-MS. This study reports proof-of-principle results of the development of an RPLC × RPLC-HRMS method using parallel gradients (2D flow rate of 0.7 mL min-1) and its comparison to shifted gradient methods (2D of 1.4 mL min-1) for the analysis of complex digests using HRMS (QExactive-Plus MS). Shifted and parallel gradients resulted in high surface coverage (SC) and effective peak capacity (SC of 0.6226 and 0.7439 and effective peak capacity of 779 and 757 in 60 min). When applied to a cell line digest sample, parallel gradients allowed higher sensitivity (e.g., average MS intensity increased by a factor of 3), allowing for a higher number of identifications (e.g., about 2600 vs 3900 peptides). In addition, reducing the modulation time to 10 s significantly increased the number of MS/MS events that could be performed. When compared to a 1D-RPLC method, parallel RPLC × RPLC-HRMS methods offered a higher separation performance (FHWH from 0.12 to 0.018 min) with limited sensitivity losses resulting in an increase of analyte identifications (e.g., about 6000 vs 7000 peptides and 1500 vs 1990 proteins).
Sujet(s)

Texte intégral: 1 Collection: 01-internacional Base de données: MEDLINE Sujet principal: Spectrométrie de masse / Protéines Limites: Humans Langue: En Journal: Anal Chem Année: 2024 Type de document: Article Pays d'affiliation: Pays-Bas

Texte intégral: 1 Collection: 01-internacional Base de données: MEDLINE Sujet principal: Spectrométrie de masse / Protéines Limites: Humans Langue: En Journal: Anal Chem Année: 2024 Type de document: Article Pays d'affiliation: Pays-Bas