The iDIA-QC v2 project aims to expand upon the first version of iDIA-QC by building a broader, more representative machine learning model for quality control (QC) in DIA-based proteomics. Specifically, we seek to:

Establish a standardized, cross-platform, high-throughput workflow for quality control of DIA-MS data.

Evaluate the reproducibility and sensitivity of both short (8–30 min) and long (45–60 min) LC gradients.

Benchmark performance across major mass spectrometry platforms, including the TripleTOF , Orbitrap, and timsTOF series.

Further validate the performance of the iDIA-QC machine learning model based on 15 key LC-MS features using standardized mouse liver digest samples.

2.1 Sample Description

We will provide all participants with trypsin-digested peptides from C57BL/6J mouse liver tissue, lyophilized and pre-aliquoted (100 µg per tube). These samples are prepared using a traditional lysis and digestion workflow to ensure consistency and batch reproducibility.

2.2 Sample Distribution

Upon completion of participant registration and receipt of signed consent to participate in the iDIA-QC v2 project, the lyophilized mouse liver digest samples will be distributed to each participant. The samples will be shipped under controlled low-temperature conditions (dry ice or equivalent) to preserve sample integrity during transit.

Each shipment will include detailed documentation outlining the peptide reconstitution protocol, storage recommendations, and handling instructions to ensure consistency across all participating laboratories. Participants are advised to store the lyophilized peptides at -80°C upon receipt and to follow the provided reconstitution procedure precisely before use in DIA-MS experiments.

The project team will coordinate closely with participants to confirm delivery and address any sample-related inquiries to maintain uniform sample processing and data quality standards.

3.1 Recommended Instrumentation

To ensure comparability across datasets, please adhere to standardized LC-MS settings for your specific instrument. The iDIA-QC v2 project currently supports:

Orbitrap series: QE, QE Plus, QE HF, QE HF-X, Exploris 120/240/480, Orbitrap Eclipse Tribrid, Orbitrap Fusion Lumos, Astral

timsTOF series: timsTOF Pro, timsTOF Pro 2, timsTOF HT, timsTOF Ultra 2

TripleTOF series: 5600+, 6600+

Detailed acquisition settings (gradient length, DIA method, injection amount) will be provided upon registration.

4.1 Registration

If you are interested in participating, please first complete the iDIA-QC v2 Registration Form. After submitting the form, an automatic email will be sent to guotiannan@westlake.edu.cn and gaohuanhuan@westlake.edu.cn. We will respond within 24 hours upon receiving the email.

4.2 Sample Distribution

After confirmation, we will coordinate the shipment of standardized mouse liver digest samples to your lab, along with a detailed protocol.

4.3 Data Acquisition and Submission

Follow the standardized LC-MS method and sample preparation protocol.

Acquire DIA-MS files according to the specified gradients (both short and long runs).

Submit raw data files along with metadata (instrument, gradient, date, LC-MS settings) to our secure data portal.

4.4 Data Evaluation and Integration

Your data will be processed using DIA-NN and the iDIA-QC pipeline.

Results will be used to retrain and refine the iDIA-QC machine learning model across platforms.

Contributing labs will be acknowledged in any resulting publications or software releases.

If you have any questions, please reach out to:

guotiannan@westlake.edu.cn, gaohuanhuan@westlake.eu.cn