Multicentre Testing Initiative (MTI) Study 2 (Vendor) – Quantitative Proteomics using data-independent acquisition mass spectrometry

Please note: In addition to the $150 participation fee, there is an additional $7 fee charged by Humantix to provide the registration and payment portal.

Context and Background

The Multicentre Testing Initiative (MTI) aims to examine reproducibility across participating facilities as well as to identify optimal strategies and workflows for specific research questions and/or sample types. To achieve this, a series of well-defined, independent studies will be conducted across participating facilities that target various mass spectrometric areas and techniques. As such, the MTI will provide participants with an opportunity to anonymously evaluate their in-house workflows in comparison to others.

Goal of this Study

This study will focus on strategies and workflows to determine relative proteomic differences

across two samples using data independent acquisition (DIA, also known as SWATH and MSe) mass spectrometry.

Study Coordinators

Matt Padula

Mark Condina

Ralf Schittenhelm

Ben Crossett

Timeline

13th Aug ‘25    Registration opens

31st Aug ‘25    Registration closes

31st Dec ‘25       Deadline for submitting results and/or raw data files

Early May ‘26    Preliminary analysis releases.  

Sample information

There will be two study samples labelled A (37 C) and B (42 C) containing complex peptide mixture derived from an E. coli digest that have been prepared by Matt Padula. Each participant will receive a pair of vials  and each vial will contain approx. 25 ug peptides.

The samples have been lyophilised and will be shipped at ambient temperature, participants should freeze samples upon arrival.

Data acquisition

Each sample has to be analysed in technical triplicates (n=3) and thus, a full dataset consists of 6 individual runs (3x sample A; 3x sample B).

Any hardware setup capable of LC-MS/MS analyses and any workflow/method can be used, but the following restrictions apply:

• 1) The mass spectrometer must be operated in data-independent acquisition (DIA) mode.

• 2) Every participant must do one set of 6 injections with a 200ng load and a 60 min linear gradient (i.e. excluding column equilibration/washing time which may make the total run time longer).  Please mark this data as “200ng-60min” in the first part of the file name.

• 3) Please try a range of linear gradients up to a maximum of 120 min.

• 4) No pre-fractionation approaches of the samples are allowed prior to the LC injection. Gas phase fractionation such as ion mobility is allowed.

• 5) The injected amount per replicate is capped at 2 ug.

Participants are encouraged to analyse the samples on as many mass spectrometers with as many methods as possible.

Data Provision

Participants should provide all acquired mass spectrometric raw / files to Matt Padula who will act as custodian of these files. A ‘raw file submission template’, which will be provided to all participants, has to be submitted alongside the mass spectrometric raw files to collect associated metadata. The deadline for returning the raw files will be approx. 2-3 months after the samples have been sent out to the participants.

All submitted mass spectrometric raw files will be de-identified by the custodian prior to further usage.

Data analysis and Optional Deliverables

De-identified mass spectrometric raw files will be forwarded to Mass Dynamics (MD) for analysis using their standardised, automated pipeline. This service will create for each dataset a comprehensive interactive analysis including numerous interactive plots. These analysed data will be provided to the Study Coordinators for further comparative analyses. In addition, each participant will get a free MD account to be able to assess their datasets independently, comparatively and anonymously.

Participants are also encouraged to analyse their mass spectrometric raw files themselves using any bioinformatic pipeline(s) they see fit. Multiple bioinformatic pipelines can be used on the same dataset. A fasta database will be provided for that purpose and the results of these in-house analyses should be sent to Matt Padula using a ‘data analysis reporting template’ (Excel), which will be provided to all participants.

Data storage and security

All files will be stored in accordance with MPMF's ISO9001 accreditation and MD’s terms of use (plain English version here). They will not be used for any purposes other than specified in this document, and they will not be put into any publicly accessible domain without written consent.

Results Compilation and Report Dissemination

Analysed data from both individual facilities and MD will be compiled and further comparatively analysed by the Study Coordinators who will present the de-identified findings at the next Australasian Core Facilities Meeting (ACFM) in 2026. Prior to the next ACFM, Personalised and de-identified reports will be sent out to all participants.