Rapidly advance quantitative proteomics with a high-throughput SWATH® Acquisition solution 

Microflow LC with OptiFlow® Turbo V Source on the TripleTOF® 6600+ System and cloud processing

Alexandra Antonoplis, Christie Hunter


To handle the increased numbers of proteomic samples required in today's biomarker research studies, SWATH Acquisition on the TripleTOF 6600+ System with the OptiFlow Source is shown to provide accurate protein quantitation results when running shorter LC gradients. The numbers of proteins quantified and the measured fold change is very similar between the 20 and 45 min gradients, as determined using the OneOmics™ Suite in SCIEX Cloud Platform. This accelerated run time per sample greatly reduces the LC-MS time required to complete a study, while maintaining results fidelity.




The combination of microflow LC with SWATH Acquisition for accelerating quantitative proteomics studies is becoming increasingly more widespread, due to the improved robustness and throughput obtained relative to the traditional nanoflow LC approach. Previously an investigation was done to characterize the tradeoff between sensitivity and throughput/robustness when switching from nanoflow to microflow for global protein quantitation experiment using SWATH Acquisition.1 It was demonstrated that with only 4x more total protein on column similar results could be achieved with much higher throughput and robustness.

The impact of shortening microflow gradient length on the number of proteins/peptides quantified from a SWATH Acquisition dataset was also studied, providing a good guidance for researchers when selecting the optimal chromatographic strategy for a study.2  

Due to these method optimizations, microflow SWATH Acquisition datasets can be generated with high throughput (up to 100 samples per day, depending on the selected gradient length).2 This will put high pressure on the downstream data processing tools and results generation, to keep pace with the generation of datasets. Thus, processing of Omics datasets in the cloud is becoming increasingly important, to handle the speed and scale of today’s industrialized proteomics approaches. 

In this work, using fast microflow gradients, a small set of cell lines were studied to further characterize the impact of fast gradients on quantitative accuracy. Data was collected using the OptiFlow Ion Source on the TripleTOF 6600+ System at two different gradient lengths, 20 and 45 mins. Using the tools in the OneOmics™ Suite in SCIEX Cloud, protein quantitation data was compared (Figure 1).

Figure 1. Differential proteins found between 20 and 45 min gradients.  Focusing on two of the cell lines in the study, HEK vs HEK_MG132 cell lines, 235 proteins were found to be differentially expressed by both gradient lengths and similar numbers of proteins were quantified (Table 1). The measured fold changes were compared to assess quantitative accuracy (Figure 3).

Key features for industrialized quantitative proteomics

  • TripleTOF 6600+ System - High speed MS/MS enables the use of 100 variable sized Q1 window isolation1 for SWATH Acquisition, for improved data quality through increased specificity, even at fastest gradient speeds 2
  • OptiFlow™ Interface 3 - Universal interface to switch between nanoflow and high flow sources in minutes
  • OptiFlow Turbo V Source 4 - Single source for all low flow LC-MS experiments with enhanced usability and robustness  
  • NanoLC™ 425 System 5 – A versatile LC system that enables the researcher to easily switch between nanoflow to high microflow LC
  • OneOmics Suite in SCIEX Cloud - a full data processing pipeline for SWATH Acquisition studies 6