Microflow SWATH® Acquisition for industrialized quantitative proteomics
NanoLC™ 400 System with TripleTOF® 6600 System
Christie L Hunter1, Nick Morrice2
1SCIEX, USA, 2SCIEX, UK
Data independent acquisition (DIA) strategies have been used to increase the comprehensiveness of data collection while maintaining high quantitative reproducibility. In DIA, larger width Q1 windows are stepped across the mass range in an LC timescale, transmitting populations of peptides for fragmentation, and high resolution MS and MS/MS spectra are acquired. Many labs are now using DIA to perform larger scale quantitative proteomic experiments with solid reproducibility on 1000s of proteins in complex matrices. As this technique increasingly proves to be a solid tool for biomarker research, larger sample sets are being analyzed, driving the need for workflow improvements that provide more throughput and higher robustness.
Nanoflow LC can provide high sensitivity and high quality separations for quantitative proteomics however the technique is relatively low throughput. Microflow LC provides higher throughput with a small loss in overall workflow sensitivity3. Here, microflow LC was investigated in combination with SWATH Acquisition on a complex matrix, to assess depth of coverage and robustness relative to current nanoflow strategies.
Chromatography: Separation of a trypsin digest of HEK cell lysate5 was performed on a NanoLC™ 425 System (SCIEX) operating in direct injection mode at microflow rates. A 0.3x150 cm ChromXP™ column (SCIEX) was used with a short gradient (4-32% solvent B in 43 min, B: 95% ACN , 0.1 formic acid in water) at 5 µL/min (total run time 57min, Figure 2). Total protein injected on column ranged from 1 – 8 µg.
Mass spectrometry: The MS analysis was performed on a TripleTOF 6600 System (SCIEX) using a Turbo V™ Source with a 25 μm I.D. hybrid electrodes (SCIEX). Variable window SWATH Acquisition methods were built using Analyst® TF Software 1.7.
Data processing: Replicate injections of each acquisition condition were processed using SWATH Acquisition microapp 2.0 in PeakView® Software 2.2 using the Pan Human SWATH Ion Library4. Modified and shared peptides were specifically excluded from quantitation. Results analysis was performed in Excel using the SWATH Replicates template. All protein and peptide numbers reported were determined at <1%FDR and <20% CV across the 5 SWATH Acquisition replicates collected.
Robustness of microflow LC
By moving up in flowrate, from 300 nL/min to 5 µL/min, a significant improvement in ease of operation is achieved. Flow path connections are more straightforward to make, and any leaks or dead volumes are more readily found and fixed. Instead of using the NanoSpray® Source for ionization, a high flow source (either Turbo V™ or DuoSpray® Source) can be used in combination with the low flow hybrid electrodes to reduce the post-column dead volumes. Higher flow rates allow faster load and re-equilibration times (Figure 2).
Very solid robustness in retention time is also observed when running microflow LC, even when running complex samples. To illustrate, a set of 48 plasma samples were running over the course of 2.5 days and the retention times of a set of dosed synthetic peptides was monitored. Excellent retention time stability was observed with deviations of 10sec or less across the gradient (Figure 3).
Optimizing SWATH® Acquisition for microflow LC
There are two modes of MS/MS acquisition on TripleTOF® Systems, the high sensitivity mode (HS >15000 resolution) and the high resolution mode (HR >30000 resolution). When processing SWATH Acquisition data, XICs of the fragment ions are generated and the width of the XIC is optimized according to the spectral peak resolution. Extraction widths of 40 and 75 ppm were used for HR mode and HS mode, respectively (settings which should extract ~80% of the spectral peak area).
Small gains in quantified proteins were observed when high resolution MS/MS mode was used on the two TripleTOF 6600 Systems tested (Figure 4).
In previous work, the sensitivity differences between different flow rates and columns was evaluated by measuring LLOQs on the QTRAP® 5500 System on a variety of peptides3. The sensitivity loss for moving from nanoflow rates on a 75 µm column to 5 µL/min on a 300 µm column was found to be ~3-4 fold. Therefore, a range of sample loads were explored to compensate for this. Typical nanoflow LC sample loads are 1-2 µg total protein on column, so amounts were increased from there (Figure 5). A steady gain in the number of proteins that could be quantified from a sample increased steadily as the load increased. Multiple human cell line digests on multiple instruments were tested to confirm this observation.
In addition to high retention time robustness, microflow LC also has very good peak shape and separation. For the 1 hour run time studied here, typical peak widths at half height were 10-12 secs. Previous work has shown that using more narrow variable width Q1 windows can improve peptide detection and increase sample coverage1,2. Using a 6 µg sample load, 5 replicates were run using 60, 80 and 100 variable windows, with a 40, 30 and 23 25 msec accumulation time respectively to maintain constant cycle time. Again, a steady gain in the number of proteins (8%) and peptides (17%) reproducibly quantified was observed as the # of windows increased.
The optimized conditions for this 1 hour total run time, for the conditions explored here, was to use a sample load of 8 µg of total protein on column, to use the 100 variable windows with a 25 msec accumulation time and to operate in the high resolution MS/MS mode. Very high quality data was obtained as shown in Figure 7, with 4963 proteins and 30685 peptides quantified at <1% FDR and <20% CV. From the cumulative %CV plot (Figure 7, top, orange line), ~90% of the peptides were quantified with <20% CV. In addition, the dynamic range of the fragment peak areas covered ~4 orders of dynamic range.
Finally, the results from this study can be compared to data collected previously on the same HEK cell digest using nanoflow LC, in order to understand when to employ the different LC strategies (Figure 8). Microflow provides much higher throughput (up to 400%), and when more material is available (4x more), data for proteins quantified can be obtained that is within 85% of what would have been obtained using nanoflow LC.
SWATH Acquisition coupled with microflow chromatography provides additional workflow options to researchers with higher throughput and robustness needs.
- Demonstrated throughput enabling ~150 proteomes per week
- 1 hour total run time for up to 24 samples / day
- Quantified 4500-5000 proteins with CV <20%
- SWATH Acquisition provides robust high quality quantitation
- Specificity key element for peptide detection and quantitation
- 80 to 100 variable Q1 windows provides increased peptide detection
- Higher resolution MS/MS (>30000 resolution) also provides increased detection using TripleTOF® 6600 System