An Automated Pipeline to Monitor System Performance in Liquid Chromatography-Tandem Mass Spectrometry Proteomic Experiments

Abstract

We report the development of a completely automated pipeline to monitor system suitability in bottom-up proteomic experiments. LC-MS/MS runs are automatically imported into Skyline and multiple identification-free metrics are extracted from targeted peptides. These data are then uploaded to the Panorama Skyline document repository where metrics can be viewed in a web-based interface using powerful process control techniques, including Levey-Jennings and Pareto plots. The interface is versatile and takes user input, which allows the user significant control over the visualization of the data. The pipeline is vendor and instrument-type neutral, supports multiple acquisition techniques (e.g., MS 1 filtering, data-independent acquisition, parallel reaction monitoring, and selected reaction monitoring), can track performance of multiple instruments, and requires no manual intervention aside from initial setup. Data can be viewed from any computer with Internet access and a web browser, facilitating sharing of QC data between researchers. Herein, we describe the use of this pipeline, termed Panorama AutoQC, to evaluate LC-MS/MS performance in a range of scenarios including identification of suboptimal instrument performance, evaluation of ultrahigh pressure chromatography, and identification of the major sources of variation throughout years of peptide data collection.

Keywords: control charts; process control; proteomics; system suitability; tandem mass spectrometry.

Figure 1

A) Overall design of the Panorama AutoQC pipeline. A Skyline template file is first created which targets a number of well characterized peptides to monitor for system suitability. An experiment is designed with appropriate controls and peptide QC data is automatically uploaded to Panorama where performance can be assessed. B) Panorama AutoQC Loader settings require the path to a Skyline template document where QC files will be imported and the folder where QC runs will be acquired, along with the type of instrument used to acquire the data. C) Panorama settings require the URL of the Panorama server and the path to a folder on the server where data will be uploaded. Also required are the login credentials of a user who has permissions to access the folder on Panorama.

Figure 2

A screenshot of the user interface for viewing QC data in Panorama A) The main toolbar with navigational links. B) Accepts user input to modify output in the displayed. C) Levey Jenning plots. D) Summary of uploaded QC data on multiple instruments.

Figure 3

Early detection of suboptimal performance. The ratio of the transition to precursor peak area is shown over 6 months of data collection for two representative peptides. A significant downward trend was observed and preventative maintenance was performed followed by a subsequent increase to values above the average. The mean of the guide set, 1 sd, 2 sd, and 3 sd are represented by the gray, green, blue, and red lines, respectively. The figure is a representative screenshot of the true image displayed by Panorama; however, axes and labels were modified to improve readability.

Figure 4

A control chart of fwhm (min) of 6 selected peptides. An annotation was created to note the switch to a small diameter particle (1.9 μm) in the control chart displaying fhwm. Another annotation noted the switch back to the standard size particle diameter (3 μm) used in our laboratories. The figure is a representative screenshot of the true image displayed by Panorama; however, axes and labels were modified to improve readability.

Figure 5

Assessment of longitudinal performance of a single peptide in terms of A) RT; B) peak area; and C) fwhm. D) Pareto analysis identifying the major sources of variation over 28 months of data acquisition in proteomic type nanoLC tandem mass spectrometry experiments. T Area = sum of integrated areas of all transitions. P Area = sum of integrated areas of precursors (M, M+1, and M+2). The mean of the guide set, 1 sd, 2 sd, and 3 sd are represented by the gray, green, blue, and red lines, respectively. The figure is a representative screenshot of the true image displayed by Panorama; however, axes and labels were modified to improve readability.