Methods

Sample preparation: Whole cell lysates (HEK293, Hela, MCF-7, A549 and MG132 treated Hela and MG132 treated HEK293) were obtained as standard cell lysates (Rockland Inc, USA).  Each cell lysate was cleaned using S-Trap MS Sample Prep Kit (Protifi, USA), then digested with trypsin using standard protocols.⁷ Sample loading of 3-5 µg of total protein (estimated) were used for each injection.

Chromatography: A NanoLC 425 System plumbed for microflow chromatography (5 µL/min) was used and operated in trap/elute mode. The analytical column used was a 0.3x150 Phenomenex Omega Polar column. Column temperature was controlled at 30°C. Gradients of 20, and 45 minutes were used, more information on gradients used can be found in previous work.²

Mass spectrometry: All data was acquired using a TripleTOF 6600+ System using the OptiFlow Turbo V Source equipped with the microflow probe and 25 µm electrodes. SWATH Acquisition data were collected using a TOF MS scan of 150 msec and 100 variable Q1 windows/cycle.⁸ Each sample was acquired in triplicate at both gradient lengths.

Data processing: SWATH Acquisition data were processed using the OneOmics Suite in SCIEX Cloud. The ion library used for processing was the Pan Human Library.⁹ Results were evaluated using the visualization applications in OneOmics Suite.⁶

Proteins quantified

SWATH Acquisition data were acquired in triplicate for all of the six cell lines, for both the 20 and 45 min gradients using microflow chromatography. With the OptiFlow source, the microflow column is attached directly on top of the microflow probe, minimizing post-column delays and dispersion for high-quality chromatography.

All the data were uploaded to the SCIEX Cloud and processed using the tools in OneOmics. After data extraction and normalization, the numbers of proteins quantified in each cell line was determined using the Analytics application, filtering the data at <1% FDR and <20% CV (Table 1). The numbers of proteins quantified using the 20 min gradient were very similar to that obtained with the 45 min gradient, although more peptides per protein were successfully quantified with the longer run time.

The MS data quality can also be assessed in Analytics (Figure 2). Visuals showing the reproducibility of replicates and the quality of the retention time alignment step can all be easily visualized. In the Browser application, the quality of the normalization step can be assessed by viewing the pre and post-normalization ratio histograms as well as the pre and post-normalization score plots.

In the Browser application, the differential proteins can be viewed in heat map form (Figure 2E) to allow investigation of specific proteins across the samples.

Quantitative accuracy of fast gradients

To determine whether accurate protein quantitation is impacted when accelerated gradients are used, the protein quantitation ratios between two cell lines (MG132 treated HEK293 vs HEK293) were compared. The MarkerView™ App in SCIEX Cloud was used to align the two datasets, and the aligned dataset was filtered to include only the proteins that shows a 2-fold fold change and 80% confidence in fold change. Principal component analysis was performed and the protein results separated into two clusters (Figure 3). The fold change values for each cluster was plotted to assess if similar results were found between the two gradient conditions. Very good correlation was observed between the protein fold changes measured with each approach, suggesting that the quantitation results are not significantly degraded when using the accelerated microflow gradients with SWATH Acquisition

Browsing results

There are a large variety of views that can be used to further delve into the biological results. One informative view is to select a cluster from the PCA analysis in the MarkerView App, then view it as a force directed graph (Figure 4). Here the proteins and their enriched gene ontologies (GO terms) are plotted together to show the relationships. Orange and blue circles show up and down-regulation of proteins and the increased depth of color shows a larger fold change. The same coloring is used for the ontologies squares. The size of the circle or square depicts the number of associations. Clicking on each will pull up a side bar that provides detailed information.

One can also look at proteins individually in the Browser app (Figure 5), here the expression differences of one protein are viewed across the 6 samples. Examples of some of the views are the Box and Whisker plot showing the protein changes. The area of the individual quantified peptides for the protein can also be viewed. 

The enriched biochemical pathways present in the data set can be viewed with the Pathways application. Pathways uses a MarkerView App results session to map clusters of compounds onto pathways present in Reactome, a peer-reviewed and open source pathways database. Upon selecting a particular cluster, the associated compounds will be mapped onto pathway diagrams in the Pathways Diagram viewer. More information can be obtained by clicking the nodes in each pathway (Figure 6). 

Conclusions

As the number of samples to be analyzed in the biological samples set steadily increases, so will the need for more robust and higher throughput protein quantitation strategies. Here, SWATH Acquisition on the TripleTOF 6600+ System with the OptiFlow Source is shown to provide accurate quantitation results when running shorter LC gradients.

• The numbers of proteins quantified is very similar between the 20 and 45 min gradients
• For the differentially expressed proteins, the fold change measured is also very similar showing quantitative accuracy is maintained in these faster experiments
• The OneOmics Suite  in the SCIEX Cloud enable fast data processing and easy results interrogation for biological insight.

References

1. Microflow SWATH Acquisition for industrialized quantitative proteomics, SCIEX technical note RUO-MKT-02-3637-A.
2. Accelerating SWATH Acquisition for protein quantitation – up to 100 samples per day - microflow LC with TripleTOF 6600 System. SCIEX technical note RUO-MKT-02-8432-A.
3. OptiFlow™ Interface for TripleTOF 6600 System - switch from nanoflow LC to microflow LC in minutes. SCIEX technical note RUO-MKT-02-7219-A.
4. Single source solution for low flow chromatography - OptiFlow Turbo V Source – always working in the sweet spot of sensitivity and robustness. SCIEX technical note RUO-MKT-02-9701-A.
5. Achieving very high reproducibility for quantitative proteomics with nanoflow LC-MS - NanoLC™ 400 System. SCIEX technical note RUO-MKT-02-5755-A.
6. Fast-track SWATH Acquisition data processing with the SCIEX Cloud - overview of the OneOmics™ Suite. SCIEX technical note RUO-MKT-02-6969-B.
7. In-solution protein digestion for proteomic samples - protocol. SCIEX technical Note RUO-MKT-02-5438-A.
8. SWATH Performance Kit - Standard Operating Protocol. 
9. Rosenberger G et al. (2014) A repository of assays to quantify 10,000 human proteins by SWATH-MS. Scientific Data, 1, 140031.

Related content

1. Sun R, Hunter CL, et al (2020) Accelerated Protein Biomarker Discovery from FFPE Tissue Samples Using Single-Shot, Short Gradient Microflow SWATH MS. J. Proteome Res. 19(7), 2732–2741.