Current targeted assays often struggle to scale to hundreds or thousands of targets. To address this challenge, we utilized the Thermo Scientific™ Stellar™ mass spectrometer in combination with Skyline software to develop large-scale targeted peptide assays. Within 2-3 days, we created a multiplex targeted proteomics method using PQ500 peptides, incorporating an adaptive RT function for the quantitation of potential protein biomarkers in plasma from patients with various diseases.
Disease and healthy plasma were purchased from BioIVT and digested using Thermo Scientific™ AccelerOme™. A Thermo Scientific™ Vanquish™ Neo UHPLC coupled with Stellar™ mass spectrometer scheme was used. Mobile phase A was 0.1% formic acid (FA) in H2O and mobile phase B was 0.1% FA in 80% acetonitrile (ACN). Thermo Scientific™ EASY-Spray™ ES906 column temperature was set at 55 ℃ and autosampler temperature was 7℃. Peptides were analyzed using a 33-minute gradient. Mass spectrometer parameters such as AGC values and maximum injection time were optimized.
The large-scale multiplexed targeted PRM method demonstrated excellent precision, linearity, and sensitivity. Utilizing an adaptive RT function and a 0.65-minute scheduled RT window, over 1,600 peptide precursors were analyzed using MS2 and MS3 assays with a 30-minute gradient, eliminating the need to reschedule retention time windows during analysis. A total of 322 endogenous proteins and 507 peptides were identified in both disease and healthy plasma using the targeted MS2 and MS3 methods. The MS3 assay enhanced the signal-to-noise ratio for low-abundant peptides or those with interference, resulting in the identification of 10.3% more proteins. Among the detected proteins, approximately 57 are FDA-approved biomarkers for various diseases. Significantly altered proteins were found to influence colorectal cancer progression.
In conclusion, a large-scale multiplexed targeted proteomics assay with adaptive RT was successfully developed to quantify potential protein biomarkers in diseased patient plasma.