Quality control of nano‐LC‐MS systems using stable isotope‐coded peptides

Abstract

In analytical sciences, there is a general need for quality control to assess whether a product or a process meets defined requirements. Especially in proteomics, which implies analysis of ten thousands of analytes within a complex mixture, quality control to validate LC-MS performance and method setup is inevitable to achieve day-to-day-, inter-system-, as well as inter-user reproducibility. Thus, results deriving from LC-MS analyses can be benchmarked and the need for system maintenance can be revealed. In particular with the advent of label-free quantification of peptides and proteins, which above all depends on highly stable and reproducible LC separations, HPLC performance has to be appropriately monitored throughout the entire analytical procedure to assure quality and validity of the obtained data. Oftentimes, proteolytic digests of standard proteins are used in this context; however, this approach implies some limitations, such as inadequate batch-to-batch reproducibility, limited (if any) dynamic range and compositional inflexibility. Here, we present an alternative strategy of nano-LC-MS/MS quality control based on a mixture of synthetic peptides covering the entire LC-gradient as well as a dynamic range of more than two orders of magnitude. Thus, (i) reproducibility of LC separation, (ii) MS performance (including limit of detection, identification and quantification), as well as (iii) overall nano-LC-MS system performance and reproducibility can be routinely monitored even in highly complex samples.

Design, Implementation and Multisite Evaluation of a System Suitability Protocol for the Quantitative Assessment of Instrument Performance in Liquid Chromatography-Multiple Reaction Monitoring-MS (LC-MRM-MS)

“…Recently, measures have been proposed to quantitatively monitor aspects of discovery-based proteomics approaches to better understand technical variability associated with chromatography, dynamic sampling, ion source configuration, signal intensity of MS and MS/MS scans, and peptide identification for data-dependent HPLC-MS/MS acquisitions (23)(24)(25)(26). Likewise, there is a critical need for standardized methods to demonstrate that LC-SID-MRM-MS analysis plat-forms are performing optimally.…”

What Can Proteomics Tell Us About Platelets?

“…However, especially when PTM-directed antibodies are used for the analysis of ubiquitination 33 or acetylation, 34 sensitivity and consequently large sample amounts can be an issue, even requiring several milligrams per sample for a comprehensive analysis. the generated data, it is important to monitor the entire workflow including (1) patient selection, (2) blood drawing, (3) platelet isolation and purity, (4) lysis and proteolytic digestion, 21 along with analytic steps such as (5) chemical labeling, 48 (6) prefractionation of the peptide mixture, (7) LC-MS analysis, 49 and (8) MS data interpretation. At present, costs and effort to conduct LC-MS-based proteome-wide analyses from individual patients are relatively high.…”

Tyr728 in the Kinase Domain of the Murine Kinase Suppressor of RAS 1 Regulates Binding and Activation of the Mitogen-activated Protein Kinase Kinase

“…Peptides were loaded in 0.1% TFA on a C18 trapping column (Acclaim Pepmap RSLC, 100 m ϫ 2 cm; Thermo Scientific) and separated on a C18 main column (Acclaim Pepmap RSLC, 75 m ϫ 15 cm) using a binary gradient (A: 0.1% formic acid; B: 0.1% formic acid, 84% acetonitrile) ranging from 5 to 50% B in 49 min, at a flow rate of 300 nl/min. Dedicated wash blanks were introduced between consecutive samples to eliminate memory effects (39). MS survey scans were acquired in the Orbitrap from m/z 300 to 2000 at a resolution of 60,000 using the polysiloxane m/z 445.120030 as lock mass (40).…”