High-Throughput Proteomics Quantification Enabled by Fast LC Separation and Advanced PRM Acquisition

Abstract

Purpose: High-throughput LC-PRM set-ups were developed, based on fast LC separations and advanced PRM acquisition schemes, to support proteomics studies. They were applied to the monitoring of the main protein components of AKT/mTOR signaling pathway enriched or not through multiplex immunoprecipitation (targeting their "total" or "phosphorylated" forms). A novel IS-PRM method variant was also developed to further increase the analytical throughput and open the way to fast broad-coverage/multi-pathway monitoring studies.

Methods: The analyses were performed on a Thermo Scientific™ Q Exactive™ HF-X hybrid quadrupole-Orbitrap™ mass spectrometer and a Thermo Scientific™ Evosep™ HF hybrid quadrupole-Orbitrap mass spectrometer operated with several PRM-based acquisition schemes (using instrument programming interface in some cases). Chromatographic separations were carried out using an Evosep One system and a Thermo Scientific™ µm3000 RSLC system equipped with capillary flow. Various gradient lengths and MS acquisition parameter settings were employed to analyze digests of high complexity (e.g., digests of human cell lines, and samples of low complexity obtained through multiplexed immunoprecipitation targeting proteins of AKT/mTOR pathway).

Results: The developed set-ups exhibited the ability to quantify with high sensitivity several dozens of endogenous peptides in one hundred samples within one day under high efficiency acquisition modes. Advanced PRM methods allowed further increases in analytical throughput without compromising the quality of quantification data when combined with multiplexed immunoprecipitation. The minor sensitivity decrease, without enrichment, for novel IS-PRM method variant relying on multiplexed isolation/fragmentation in single MS/MS spectrum acquisition, turned out to be a promising option for broad-coverage/multi-pathway monitoring studies while maintaining acceptable quantification performance.

Introduction

Targeted analyses based on HR/AM parallel reaction monitoring (PRM) measurements have opened new opportunities in quantitative proteomics. The PRM technique has delivered a significant increase in selectivity of measurements, allowing more sensitive endogenous peptide quantification in complex samples. Refined acquisition methods, e.g., internal standard triggered PRM (IS-PRM)¹, have enabled larger-scale experiments while still providing exquisite data quality. Here, the potential of PRM combined with fast capillary flow LC separation has been explored to accelerate the throughput of targeted analyses.

Materials and Methods

Sample Preparation

Cell Culture: HCT116 cells were grown in McCoy’s 5A Media with 10% FBS/1%PenStrep to ~70-80% confluency. HCT116 cells were serum starved in 0.1% charcoal stripped FBS for 24 hours prior to the following treatments: untreated, stimulated (15 min hIGF-1 (100ng/ml), Cell Signaling Technology PN89117SF). Subsequent to treatments, cells were lysed with IP-lysis buffer (Thermo Fisher Scientific PN88778B) supplemented with 1X Halt Protease and Phosphatase inhibitor cocktail (Thermo Fisher Scientific PN88668). Protein concentration of lysates was determined with BCA Protein Assay (Thermo Fisher Scientific PN23225). The Thermo Scientific™ Pierce™ MS-compatible Magnetic IP, Protein A/G (Thermo Fisher Scientific PN900409) was used to screen and validate antibodies for 13 total and 12 phosphorylated AKT/mTOR signaling pathway enriched cell lysates. Validated antibodies were biotin tagged with the Thermo Scientific™ Pierce™ EZ-Link™ Biotinylation Kit (Thermo Fisher Scientific PN21430). The Thermo Scientific™ Pierce™ MS-Compatible Magnetic IP Kit, Streptavidin (Thermo Fisher Scientific PN900408) was used to multiplex IPs for target enrichment. IP samples were processed by an in-solution digestion method in which IP eluates were reconstituted with 8M Urea, 50mM TEAB, pH 8.5 followed by reduction, alkylation and trypsin (Thermo Fisher Scientific PN90057) digestion overnight at 37°C. All samples were desalted with C18 FA.

MS sample preparation: A set of 32 high-quality Pierce™ stable isotopically labeled (SIL) peptides corresponding to 13 proteins from AKT/mTOR pathway was spiked at 20 fmol in 500 ng of HCT116, 1 mg/ml (0.1 mg/µl) with no enrichment. For the generation of the dilution series solutions in "low" complexity matrix, the set of 32 SIL peptides was spiked in various calibrated amounts (7 points from 50 amol to 200 fmol, and one matrix blank) in 1 mg of a β-globin mass digest (Thermo Fisher Scientific PN88342) supplemented with 20 fmol of synthetic unlabeled forms of the peptides. For the preparation of the dilution series solutions in "high" complexity matrix, the set of 32 SIL peptides was spiked in various calibrated amounts (7 points from 50 amol to 200 fmol, and one matrix blank) in 500 ng of a HeLa digest (Thermo Fisher Scientific PN88329) supplemented with 20 fmol of synthetic unlabeled forms of the peptides.

A set of 186 standard-quality SIL peptides (Pierce, Rockford, IL, JPT Technologies, Berlin, Germany) corresponding to 111 challenging proteins was spiked at 20 fmol (in the normal amount) in 1 mg of a HeLa digest and used to follow MAPK, WNT, RAS, ErbB and AKT/mTOR pathways in the multi-pathway monitoring experiment. All samples were supplemented with 30 fmol of a mixture of PRTC peptides (Thermo Fisher Scientific, PN88321).

LC-MS/MS Analysis

For single-signaling pathway monitoring experiments, chromatographic separations were performed on an Evosep One system (Evosep, Odense, Denmark) equipped with Evosep C30 EvoTips and C18 analytical columns (3 µm, 0.1 x 80 mm) operated at 1.2 µl/min, 0.5 µl/min or 0.15 µl/min flow rates. For multi-pathway monitoring experiments, chromatographic separations were performed on a µm3000 RSLC system (Thermo Fisher Scientific) equipped with C18 trap cartridges (5 µm, 0.3 x 5 mm) operated at 100 µl/min and analytical columns (2 µm, 0.15 x 150 mm) operated at 3 µl/min. The various gradients length are listed in relevant figures. Evosep One and Ultimate 3000 RSLC systems were coupled to Q Exactive HF-X MS or Q Exactive HF quadrupole-Orbitrap MS instruments, respectively. Mass spectrometers were operated with several PRM-based acquisition schemes including dRT-PRM, IS-PRM (using the instrument application programming interface, IAPI). Under its main implementation ("sequential"), the IS-PRM technique demonstrated to be a "watch mode" in which internal standards were first quantified and then used in the dynamically controlled elution and monitoring windows for the endogenous analytes, and a "quantitative mode" (triggered by the real-time detection of the IS by means of spectral matching), which measured the corresponding pairs of IS and endogenous peptides serially over their elution profile, using optimized acquisition parameters (Figures 1 and 2). For all PRM, dRT-PRM, and IS-PRM (Quant. mode) experiments on Q Exactive HF-X instrument, PRM scans employed an Orbitrap resolution of 60,000 at m/z 200 and maximum fill times of 116 ms. The PRM scans on µm3000 RSLC an Orbitrap resolution of 7,500 (at m/z 200) and maximum fill times of 10 ms. A variant of the IS-PRM method, relying on the simultaneous measurement of SIL and endogenous peptides by "multiplex" acquisition of their fragment ions in a single MS/MS spectrum in Quant. mode, was developed and tested (Figures 1 and 2) to further increase analytical throughput.

Results and Discussion

^a For Q Exactive HF-X MS. ^b Estimated as 5-6 and 2-3 x peak width in conventional and IS-PRM analyses, respectively. For a sampling rate of 6 data points/peak. *For a sampling rate of 6 data points/peak. **in QUANT mode

AKT/mTOR Pathway Monitoring by Fast LC-PRM

Fast LC-PRM methods were applied to the monitoring of a single signaling pathway, the main components of AKT/mTOR pathway (Figure 4, upper right panel). A total of 13 proteins were defined as main components of AKT/mTOR pathway. The Evosep LC method, allowing 60 samples/day acquisition, was selected as surrogate for the 13 AKT/mTOR proteins. The PRM assays developed for the quantification of AKT/mTOR surrogate peptides were applied to untreated and hIGF-1 stimulated HCT116 digest prepared i) by multiplex immunoprecipitation targeting phosphoproteins (Figure 4, lower right panel), ii) by multiplex immunoprecipitation targeting "total" proteins (Figure 5, upper panel), and iii) by multiplex immunoprecipitation targeting "total" proteins digest and analyzed on the LC column for conventional PRM (Figure 5, lower panel). Peptide assays characteristics (i.e. LOD, LOQ, and linearity) and the total ion chromatograms displayed in left panels of Figures 4 and 5. Peptide surrogates were quantified based on the multiplex immunoprecipitation targeting phosphoproteins in triplicate LC-PRM analyses. While hIFG-1 stimulation did not induce changes in total proteins abundance, it modified the activation status of most of them, as illustrated by the significant increase in the peptide abundance of phosphoproteins, especially IGF-1R, INSR, and AKT proteins. Multiplex immunoprecipitation steps allowed differentiated quantification of phospho- and total-proteins but also quantification of measurements of pairs of SIL and endogenous in triplicate LC-PRM analyses. The multiple immunoprecipitation steps allowed differentiated quantification of phospho- and total-proteins but also quantification of measurements of pairs of SIL and endogenous peptides corresponding to the target proteins (Table 1 and Figure 4, upper panel) and in low complexity matrix presented in Figure 4 (lower right panel). The multiple immunoprecipitation steps allowed differentiated quantification of phospho- and total-proteins but also quantification of measurements of pairs of SIL and endogenous peptides corresponding to the target proteins (Table 1 and Figure 4, upper panel) and in low complexity matrix presented in Figure 4 (lower right panel).

Throughput Capabilities of Advanced PRM Methods

AKT/mTOR pathway monitoring experiments were repeated using advanced PRM methods. The higher efficiency of the Evosep LC method, allowing 100- and 200-samples/day analytical throughput, respectively, while keeping same PRM scans settings (quant. mode for IS-PRM). The analyses exhibited sampling rates similar to those of conventional PRM analyses (approx. 60 data points/peak) (Figure 8). The compressed gradients with advanced PRM methods did not affect the sensitivity of measurements in moderately compressed or low complexity matrix (Figures 7 and 8).

Towards Higher Throughput PRM Acquisition Variant

A novel IS-PRM method variant, relying on multiplexed isolation/fragmentation of pairs of SIL and endogenous peptide precursor ions and single MS/MS spectrum acquisition, was explored. It was applied to the measurement of the dilutions series of the 32 SIL peptides surrogate of the 13 AKT/mTOR proteins in low complexity matrix using "200 samples/day" LC setup. The method exhibited good qualification accuracy (Figure 9) while enabling a 3-4 fold increase in experiment scale, as compared with sequential IS-PRM analysis. This was illustrated by the analyses of 186 pairs of SIL and endogenous peptides using "100 samples/day" LC method with satisfying sampling rate (Figure 10).

Considerations for High-Throughput LC-PRM Analysis

The chromatographic properties of the Evosep system were determined from LC-MS analyses of 15 PRTC peptides. The system was operated at throughputs of 60, 100, and 200-samples analyzed per day, allowing to define the total elution window and peptide chromatographic peak widths that can be included in conventional PRM, dRT-PRM and IS-PRM. This allowed an ideal situation where the elution times of separation, and more common situation under which peptide elution times are compressed into chromatographic run, the higher acquisition efficiency and experiment increase in scale at constant analytical throughput.

Conclusions

• The developed fast LC-PRM set-ups exhibited the ability to quantify with high sensitivity several dozens of endogenous peptides in one hundred samples within one day under high efficiency acquisition modes.
• Advanced PRM methods combined with multiplexed immunoprecipitation increased the throughput of signaling pathway monitoring without compromising data quality. The novel single-scan IS-PRM msx method turned out to be an attractive option for broad-coverage/multi-pathway monitoring studies, which however needs to be further investigated.