MS/MS ALL with SWATH Acquisition Comprehensive Quantification

By Stephen Tate and Christie Hunter (SCIEX)

In proteomics, and many other sample types, the complexity and dynamic range of compounds is very large. This poses challenges for the traditional data dependent workflows, requiring very high speed MS/MS acquisition to deeply interrogate the sample in order to both identify and quantify a broad range of analytes. Data independent acquisition strategies have been used to increase the reproducibility and comprehensiveness of data collection. However, mass spectrometers have been limited in the speed and quality of the data that they acquire. With the TripleTOF™ 5600 System, it is now possible to perform a data-independent workflow with high speed and high resolution in both MS and MS/MS modes. Targeted quantification is often desired to obtain accurate quantification in complex mixtures. Multiple Reaction Monitoring (MRM) is the most dominant workflow for performing this type of analysis, but the nominal mass selection of triple quads can allow mass interferences. There have been a number of workflow extensions that provide higher specificity to targeted quantification. On the TripleTOF™ 5600 system using the MRMHR workflow, the instrument is set up to acquire full scan MS/MS data on a fixed precursor, over and over again across the LC run (Figure 1). The Q1 is fixed, the peptide is fragmented in the collision cell and the full scan TOF MS/MS is acquired. After data acquisition, extracted ion chromatograms on sequence specific ions are generated. MS/MSALL with SWATH Acquisition brings together data independent acquisition for comprehensive acquisition and targeted quantification for the highest quality data.


Figure 1. MRM-HR Workflow Using the TripleTOF™ 5600 System. In the MRM-HR workflow, a fixed number of analytes are targeted and high resolution MS/MS spectra are collected across an LC run. Precursor masses are selected at narrow resolution such that mainly the target compound is passed into the collision cell (top). This produces a full scan MS/MS spectrum enriched for the analyte of interest (right). Then, any number of fragment ions can be extracted at high resolution post-acquisition to generate MRM-like data (bottom).

In MS/MS-ALL with SWATH Acquisition, the Q1 quadrupole is stepped at 25 amu increments across the mass range of interest, passing a 25 amu window through into the collision cell. The transmitted ions are fragmented and the resulting fragments are analyzed in the TOF MS Analyzer at high resolution. This sequential windowed acquisition is termed SWATH Acquisition (Figure 2). A large mass range can be interrogated in an LCMS time frame because of the larger mass steps. This yields a more complicated MS/MS spectrum at each step; however, the high resolution MS/MS enables tighter extraction windows to maintain high specificity. The advantages of this approach for targeted quantification are numerous. No upfront assay development is required on specific targets, all data is acquired and targets are mined post-acquisition. Data collection is comprehensive so quantitative data on additional compounds can mined retrospectively. Further, in the event of overlapping fragment ions, the quantification of any peptide or protein can be “rescued” be re-assigning quantification to a different fragment ion or peptide. In similar analyses using data-dependent or targeted MRMs, the offending peptide or protein would have to be excluded from the results. Finally, the limits of detection and quantification rival those of the leading triple quads available today.

Figure 2. MS/MS-ALL with SWATH Acquisition. In this workflow, instead of the Q1 quadrupole transmitting a narrow mass range through to the collision cell, a wider window containing more analytes is passed. This produces a more complex MS/MS spectrum which is a composite of all the analytes within that Q1 m/z window. Because the fragment ions are high resolution, high quality XICs can be generated post-acquisition to produce the MRM-like data. This Q1 window can be stepped across the mass range, collecting full scan composite MS/MS spectra at each step, with an LC compatible cycle time. This enables a data-independent LC workflow.

In one example of the workflow, a digest of a yeast cell lysate was analyzed using MS/MS-ALL with SWATH Acquisition and interrogated for a large number of proteins. In a single injection, MS/MS quantification is obtained on proteins across a broad dynamic range, down to 100s of copies per cell (Figure 3).

Figure 3. Broad Dynamic Range of Protein Quantification Possible with MS/MS-ALL using SWATH Acquisition. A yeast cell digest was analyzed using this workflow and interrogated for yeast proteins across a broad dynamic range. Depth of coverage similar to that obtained by MRM analysis was obtained but with comprehensive acquisition and highest multiplexing.


•Comprehensive quantitative analysis with qualitative confirmation

•Quantification with no method development for all species in a single analysis

•Archive of all analytes enables retrospective in silico interrogation

•High resolution quantification reduces potential for interferences

•Quantitative performance comparable to leading triple quadrupole instruments