Mar 05, 2026
Protocol for the LC-MS/MS-based proteomics assay of samples generated using the MPLEx method
- Isaac Attah1,
- Christopher Anderton1,
- Geremy Clair1
- 1Pacific Northwest National Laboratory
- Human BioMolecular Atlas Program (HuBMAP) Method Development Community
- Pacific Northwest National Laboratory (PNNL)
Protocol Citation: Isaac Attah, Christopher Anderton, Geremy Clair 2026. Protocol for the LC-MS/MS-based proteomics assay of samples generated using the MPLEx method. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld1jj9l5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 16, 2026
Last Modified: March 05, 2026
Protocol Integer ID: 243411
Keywords: Proteomics, LC-MS/MS, DIA, Tissue, HubMAP, ms proteomics data independent acquisition, based proteomic, proteomic, mplex method this protocol, mplex method, hubmap tmc lung, hubmap program, protocol for the lc
Funders Acknowledgements:
NIH - Biorepository for Investigation of Neonatal Diseases of Lung-Normal (BRINDL-NL)
Grant ID: U01HL122700
NIH - Biorepository for INvestigation of Diseases of the Lung (BRINDL) - Phase II
Grant ID: U01HL148861
NIH - The Human Lung BioMolecular Multi-Scale Atlas Program (HuBMAP-Lung)
Grant ID: U54HL165443
NIH - Research Center for Spatiotemporal Lung Imaging and Omics
Grant ID: U01HL148860
Abstract
This protocol provides details on how the LC-MS/MS proteomics Data Independent Acquisition (DIA) samples were run for the HubMAP program (HubMAP TMC Lung and Pancreas).
Materials
Thermo Scientific Orbitrap Astral
Troubleshooting
Sample Preparation
Proteins are extracted using a modified Folch extraction (Folch 1957; Nakayasu, et al., 2016) termed MPLEx. The protocol utilized is provided on the protocols.io titled:
Extracted proteins are then processed using a urea/trypsin-based method:
LC-MS/MS DIA Analysis on the Astral Instrument
5 µl is injected into the liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) system, comprising a Thermo Vanquish Neo with a Thermo Astral mass spectrometer.
Note: Vialed samples can be conserved for up to 3 months in a -20 ˚C freezer
The ESI source parameters are set as follows: spray voltage 2.2 kV in positive ionization mode; capillary temperature 300 °C; S lens RF level 40 arbitrary units.
MS1 parameters are set as follow:
- MS range 380-980 m/z
- Resolution 240K
- Automatic Gain Control (AGC) target : 5e6
The DIA mode parameters are set as follows:
- Precursor isolation window 2 m/z ranges
- MS range 380-980 m/z
- AGC target : 5e5
- HCD collision energy 25% normalized
Liquid Chromatography Parameters
The column used was a Thermo ES906 column held at a temperature of 45 °C. An injection volume of 5 uL was used.
All solvents and additives used were LC-MS grade. Mobile phase A (MPA) consisted of 100% water with 0.1% formic acid. Mobile phase B (MPB) consisted of 100% Acetonitrile with 0.1% formic acid.
The gradient used was the following
| A | B | C | D | |
| Time (min) | Flowrate (mL/min) | % MPA | % MPB | |
| 0 | 3.5 | 99 | 1 | |
| 0.001 | 2.8 | 99 | 1 | |
| 0.501 | 1.3 | 96 | 4 | |
| 0.901 | 1.1 | 92 | 8 | |
| 12.301 | 1.1 | 97.5 | 22.5 | |
| 15.301 | 1.1 | 65 | 35 | |
| 15.501 | 1.1 | 45 | 55 | |
| Column wash | ||||
| 16.001 | 2.8 | 1.0 | 99.0 | |
| 17.201 | 2.8 | 1.0 | 99.0 | |
| 18.0 | 2.8 | 99.0 | 1.0 |
QA/QC
Calibration is done using Pierce‱ FlexMix‱ Calibration Solution. Mobile phase solvents are topped off between experiment sets (i.e., not during a sample set) to reduce drifting retention times.
Blanks are run before the start of every new sample set, and QC (mammalian peptide lysate) is run systematically after every sample set.
Data Analysis
The *.raw files generated are analyzed using DIA-NN. An example of protocol is provided here:
Acknowledgements
This work was supported by the National Heart, Lung, and Blood Institute (NHLBI) Molecular Atlas of Lung Development Program Human Tissue Core (LungMAP HTC) and LungMAP BioRepository for INvestigation of Diseases of the Lung (BRINDL) through grants U01HL122700 and U01HL148861 (to GS Pryhuber), and U01HL148860 (G Clair) and by the NIH Common Fund grant U54HL165443 (to GS Pryhuber with Co-Investigators G Clair, and C Anderton). Part of this work was performed in the Environmental Molecular Science Laboratory, a U.S. Department of Energy (DOE) national scientific user facility at Pacific Northwest National Laboratory (PNNL). Battelle operates PNNL for the DOE under contract DE-AC05-76RLO01830. The opinions expressed in this article are the authors’ own and do not reflect the view of the NIH, the Department of Health and Human Services, or the U.S. government. We are very grateful for the generosity of the donor families and honor their loss.