ABSTRACT
Purpose: The hybrid-DIA method aims to comprehensively digitize a clinical specimen while simultaneously enhancing measurement sensitivity for a specific set of markers of clinical interest. Diagnostics level-1 markers (of current clinical interest; 1%) & level-2 markers (of potential clinical interest & pathways; 9%) can be accurately monitored in an absolute fashion via highly sensitive and intelligent on-the-fly triggered multiplexing PRM scans, while simultaneously discovery-driven DIA allows to establish the clinical proteotype of level-3 information (markers of currently unknown clinical value; 90%) in single hybrid-DIA MS experiment.
Methods: Experiments benchmarking standard DIA methods, PRM methods, and the novel HybridDIA methods were performed using a nanoLC system coupled to the Orbitrap Exploris 480 MS. The Hybrid-DIA method is programmed in C#, utilizing the iAPI.
Results: The preliminary data demonstrate that the peptides of interests can be detected with a higher signal-to-noise ratio, lower limit of detection and quantitation, fewer interferences, and lower CVs at lower concentration for Hybrid-DIA MS than for standard DIA MS, while simultaneously comprehensively profiling proteomes with one single shot LC-Hybrid-DIA analysis.
INTRODUCTION
Sample Preparation
SpikeTides™ Set TAA - heavy and light (JPT technologies) were resuspended in LC-LOAD(Preomics) at an approximate concentration of 10 pmol/ml. Dilutions from these stock solutions were prepared accordingly. Homemade Hela lysate was prepared digesting 100 mg of protein input material using the Preomics kit. Homemade Hela lysate was used at a concentration of 1 mg/ml as background matrix in the dilution series.
Methods
The quantitation of the TAA marker panel and the global proteome profiling performance of the hybrid-DIA MS methodology were characterized and benchmarked to the standard PRM and DIA methods. Samples were separated with a Thermo Scientific™ EASY-nLC™ 1200 system using a PepMap™ column (ES803) and analyzed with a Thermo Scientific™ Orbitrap Exploris™ 480 mass spectrometer in standard data independent acquisition (DIA), parallel reaction monitoring (PRM), and intelligent data acquisition Hybrid-DIA mode with 2hour LC gradient with a target list of 120 and 179 , respectively. Three technical replicates are analyzed per condition. The hybrid-DIA method is programmed in C#, utilizing the instrument API on an Orbitrap Exploris 480 MS.
Data Analysis
Targeted quantitation data analysis pipeline for Hybrid-DIA (msxPRM scans) is shown in Fig. 3, where the data were processed and quantified to calculate the Heavy/Light ratio using Python, Skyline, and shinnyR. The .HTRMS converter (Biognosys) was applied to extract the full scan and DIA scans from the Hybrid-DIA files into the .HTRMS files, which were then analyzed in Spectronaut™ v.15 software (Biognosys) using spectral-library free directDIA search. Standard DIA raw data files were directly analyzed in Spectronaut v.15 (Biognosys) using directDIA search. Standard PRM raw data files were analyzed using Skyline software.
RESULTS
Intelligent Data Acquisition Hybrid-DIA MS Strategy
Hybrid-DIA MS can minimize sample consumption and measurement time by merging different
acquisition schemes into one LC-MS run, reducing data missingness, enabling researchers to combine
DIA-driven discovery phase and targeted-driven validation phase in one step and in one single
experiment.
Quantitation of diagnostic markers
Improved quantitation of diagnostic markers with Hybrid-DIA
We tested the Hybrid-DIA method on a pool of 252 representative proteotypic peptides for tumor associated antigens (TAA) derived from 61 annotated human proteins. We generated mixtures containing both the heavy reference peptide as well as its synthetic light isotope. Whereas the heavy reference peptide was kept constant in all samples at approximately 100 femtomole (injected onto column), its light counterpart was measured in a dilution series ranging from 100 femtomole to 100 atomole. We monitored 179 TAA peptides at a time in a scheduled fashion using high-resolution msxPRM while at the same time recording the DIA traces in Hybrid-DIA.(Fig. 5A)
The narrow isolation window and maximized ion injection time of the msxPRM scans of Hybrid-DIA method improve the selectivity and sensitivity of quantitation, as well as the confidence of detection and quantitation, especially with high background matrix (Fig. 5). The linearity of quantitation of detected peptides were improved to 0.9995 for Hybrid-DIA method, better than the standard DIA method (0.95), and as good as standard PRM methods (0.9995) (Fig. 5B). Compared to the standard DIA method, hybrid-DIA is able to improve the LOD/LOQ and achieve better quantitation precision with lower CVs of the targeted peptides, especially for the low abundant endogenous peptides at low concentration ranges (Fig. 5C).
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