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MacCoss - DIA to SRM Assays

Targeted MS Experiment

 
DIA to inform Triple Quad Assay development
Permanent Link: https://panoramaweb.org/DIAtoSRM.url Share
Data License: CC BY 4.0 | ProteomeXchange: PXD059611 | doi: https://doi.org/10.6069/qhpr-0916
  • Organism: Homo sapiens
  • Instrument: TSQ Altis,Q Exactive HF
  • SpikeIn: No
  • Keywords: DIA, SRM, MRM, targeted proteomics, quantitative proteomics, cerebrospinal fluid, Alzheimer's disease, neurodegenerative disease
  • Lab head: Michael MacCoss Submitter: Deanna Plubell
Abstract
Mass spectrometry based targeted proteomics methods provide sensitive and high-throughput analysis of selected proteins. To develop a targeted bottom-up proteomics assay, peptides must be evaluated as proxies for the measurement of a protein or proteoform in a biological matrix. Candidate peptide selection typically relies on predetermined biochemical properties, data from semi-stochastic sampling, or by empirical measurements. These strategies require extensive testing and method refinement due to the difficulties associated with prediction of peptide response in the biological matrix of interest. Gas-phase fractionated (GPF) narrow window data-independent acquisition (DIA) aids in the development of reproducible selected reaction monitoring (SRM) assays by providing matrix-specific information on peptide detectability and quantification by mass spectrometry. To demonstrate the suitability of DIA data for selecting peptide targets, we reimplement a portion of an existing assay to measure 100 Alzheimer’s disease proteins in cerebrospinal fluid (CSF).(Spellman). Peptides were selected from GPF-DIA based on signal intensity and reproducibility. The resulting SRM assay exhibits similar quantitative precision to published data, despite the inclusion of different peptides between the assays. This workflow enables development of new assays without additional up-front data acquisition, as demonstrated through a separate assay generated for an unrelated set of proteins in CSF.
Experiment Description
We use gas phase fractionated libraries collected from biological samples to efficiently select peptides for SRM. The indexed retention time (iRT) values and transitions are inherently determined during the generation of the library, making SRM scheduling and transition selection seamless. The workflow minimizes the number of comprehensive measurements to assess which peptides 1) are good responders in the matrix of interest, 2) chromatograph well, and 3) exhibit stability and precision for measurement. To demonstrate the usefulness of this workflow we compare peptides selected from narrow-window DIA to those included in an established, well-characterized SRM assay for Alzheimer’s disease in cerebrospinal fluid for a set of proteins. Using the workflow presented an assay for the same proteins of interest was developed, exhibiting equivalent precision and accuracy but bypassing multiple rounds of selection and refinement. Additionally, the technique described is implemented to generate a separate quantitative assay for different targets from the same initial DIA data.
Sample Description
Cerebrospinal fluid (CSF) samples were obtained by lumbar tap from patients diagnosed as either probable Alzheimer’s disease or Parkinson’s disease, and healthy age-matched controls as a part of the Alzheimer’s disease resource center at the University of Washington, or the Udall repository at Stanford University. CSF was obtained following NIA AD Center Best Practices Guidelines and samples stored at −80°C. CSF samples were combined to make a pooled reference for method development. All samples were pre-existing and were collected under protocols approved by the Institutional Review Board (IRB) at University of Washington or Stanford University prior to the start of this project.
Created on 1/9/25, 7:24 PM

Targeted MS Runs

 
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Download
DIA_CSF_CombinedLibs_quant_2025-01-08_14-02-02.sky.zip
2 GB 
2025-01-09 19:22:541,1545,5955,59561,6353
20180527_SRM_results_2025-01-03_16-10-45.sky.zip
10 MB 
2025-01-09 19:22:541013243241,5833
pain_target_peptides_DIA_2025-01-03_16-09-15.sky.zip
100 MB 
2025-01-09 19:22:54704104102,1883
pain_target_SRM_2025-01-03_16-06-30.sky.zip
6 MB 
2025-01-09 19:22:54701521527713
DIA_targets_AD_2025-01-03_15-13-47.sky.zip
188 MB 
2025-01-09 19:22:54991,0001,00011,0733

Overview

 

Mass spectrometry based targeted proteomics methods provide sensitive and high-throughput analysis of selected proteins. To develop a targeted bottom-up proteomics assay, peptides must be evaluated as proxies for the measurement of a protein or proteoform in a biological matrix. Candidate peptide selection typically relies on predetermined biochemical properties, data from semi-stochastic sampling, or by empirical measurements. These strategies require extensive testing and method refinement due to the difficulties associated with prediction of peptide response in the biological matrix of interest. Gas-phase fractionated (GPF) narrow window data-independent acquisition (DIA) aids in the development of reproducible selected reaction monitoring (SRM) assays by providing matrix-specific information on peptide detectability and quantification by mass spectrometry. To demonstrate the suitability of DIA data for selecting peptide targets, we reimplement a portion of an existing assay to measure 100 Alzheimer’s disease proteins in cerebrospinal fluid (CSF).(Spellman). Peptides were selected from GPF-DIA based on signal intensity and reproducibility. The resulting SRM assay exhibits similar quantitative precision to published data, despite the inclusion of different peptides between the assays. This workflow enables development of new assays without additional up-front data acquisition, as demonstrated through a separate assay generated for an unrelated set of proteins in CSF.

 

File mapping and descriptions:

Folder Contents Description
raw_AD_SRM CSF AD-associated protein SRM assay Altis Triple Quad raw files
raw_DIA CSF QE-HF GPF-DIA raw files
raw_Pain_SRM CSF Pain-associated protein SRM assay Altis Triple Quad raw files
raw_PRTC_scheduling_SRM Pierce Retention Time Calibrant peptide SRM Altis Triple Quad raw files used for scheduling SRM assays
search_results EncyclopeDIA search result files