Here, the impact of shorter LC gradient lengths on the number of proteins that can be quantified by SWATH Acquisition using the TripleTOF 6600 System was investigated. Due to the very fast MS/MS acquisition rates possible on TripleTOF Systems, high numbers of peptides/proteins can be quantified with very fast gradients.
The SWATH® Acquisition data independent acquisition strategy provides a comprehensive quantitative analysis of complex samples, providing full scan high resolution MS and MS/MS on all detectable compounds eluting off the column. These datasets can then be re-interrogated over and over again as new information on new compounds of interest emerges. Specificity can be optimized by balancing the Q1 window width, the number of Q1 windows and accumulation time per window, such that analyte S/N can be improved significantly while maintaining good quality quantitative results.
Here, the use of several spectral libraries for accurate compound identification from accurate mass DDA and DIA workflows was compared, to evaluate the number of metabolites accurately identified in an extracted plasma sample in a single injection workflow. The LC-MS/MS libraries evaluated in this study include NIST 17, MoNA, METLIN, and SCIEX libraries.
SWATH® Acquisition, a data independent acquisition (DIA) workflow is well adopted in quantitative discovery proteomics, but still not commonly used in discovery metabolomics. Here, how SWATH® acquisition enables the identification of a higher number of metabolites for untargeted metabolomics workflows is described and compared to traditional data dependent acquisition (DDA) approaches thus enabling a broader profile of the metabolome1. Our results show that SWATH Acquisition using variable windows improves metabolite coverage using the Accurate Mass Metabolite Spectral Library (AMMSL) compared with the traditional DDA approach.
Here, the OneOmics suite was used to process a SWATH acquisition data set acquired using on the SCIEX ZenoTOF 7600 System, with the goal of quantifying differences in metabolites found in ZDF and healthy rat urine to demonstrate the workflow. The complete integration of data processing applications in the cloud-based platform enabled rapid processing of the study data. The platform features innovative algorithms and a wide range of tools for quantifying metabolites with confidence and exploring the biological relevance of the results.
Here, significant gains in the peptides and proteins quantified (332% and 118% gains, respectively) from SWATH Acquisition data are demonstrated, when the data processing is done with a large pan proteome library rather than a smaller library generated from 1 dimensional LC-MS experiments. For the cell lysate SWATH Acquisition data investigated here, 5922 proteins were quantified at <20% CV when processed using the very comprehensive pan human library. Therefore, selecting an appropriate ion library strategy for data processing that matches the depth of coverage required for the biology under study is very important.
Here microflow SWATH® Acquisition on a TripleTOF® 6600 System was used to perform quantitative protein profiling with SWATH® Acquisition on a set of 105 colon cancer samples obtained from a biobank in 5 days. High analytical depth was obtained with ~4,500 proteins quantified across the two sample types, healthy, and colon cancer.
SWATH® Acquisition with variable Q1 windows coupled with microflow chromatography provides additional workflow options to researchers with higher throughput and robustness needs. Using a total run time of 1 hour per proteome, 4500-5000 proteins can be quantified reproducibly.
Processing of DIA data in the cloud significantly accelerates time to answers. Two new algorithms involving combining ion libraries and performing retention time calibration have been developed to further streamline the SWATH Acquisition data processing pipeline in OneOmics™ Suite.