Mass spectrometer setup and data analysis

Liquid chromatography tandem mass spectrometry (LC-MS/MS) experiments were carried out at the Mass Spectrometry Core Facility at Charles Perkins Centre of the University of Sydney using high-resolution TripleTOF® 6600 Quadrupole Time-Of-Flight (QTOF) and Q ExactiveTM mass spectrometers. Samples prepared for MS with RIME or in-gel enzymatic digestion method were reconstituted in loading buffer and were loaded into mass spectrometer with 10 µL. Each sample contained peptide mixture, and was separated by liquid chromatography (LC) based on hydrophobicity and then ionised by electrospray (ESI). Mass

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spectrometer detects the intensity of ion signals as a function of retention time, as well as precursor ions and product ions of peptides based on mass-to-charge (m/z) ratios.

TripleTOF® 6600 Quadrupole Time-Of-Flight (QTOF, SCIEX) mass analyser provides high sensitivity, mass resolution and mass accuracy of tandem mass spectrometer in both precursor (MS) and product ion (MS/MS) modes (Morris et al., 1996, Shevchenko et al., 1997). It has been widely used as a powerful and robust tool with unique capabilities. The QTOF system has a series of quadrupole filters that transmit ions according to their mass-to-charge (m/z) value. The first quadrupole is the QJet® ion guide. It is located between the orifice plate and the Q0 region, and it doesn’t filter the ions generated by ionisation but focuses them before they enter Q0. In Q0 region, ions are focused again before passing into Q1. The pre-focus of large ion flux enhances the sensitivity of the instrument and improves signal-to-noise ratio. The Q1 quadrupole filters the ions before they enter Q2 quadrupole. The Q1 quadrupole can pass all ions within a specified m/z range to the Q2 collision cell, known as a TOF MS scan and all ions are analysed by the TOF system; or it can only pass one ion with a specified m/z ratio to the Q2 collision cell, known as a TOF MS/MS scan, and only the selected ion is analysed. Within the Q2 collision cell, the internal energy of parent ions is increased by collisions with gas molecules to break the molecular bonds and thus generating product ions. After passing through the Q2 collision cell, the ions enter the TOF region for additional mass analysis. Due to the different m/z ratio, each ion reaches the detector at different time and generates a current that is subsequently converted into a voltage pulse. The number of pulses depends on the quantity of ions entering the detector. The mass spectrometer converts the voltage pulses to a signal and then correlates the signal to the time it takes each ion to reach the detector. The signal reflects the ion intensity and the time to reach the detector represents a specific m/z value. The mass spectrometer displays this data as a mass spectrum, including spectra of TIC (total ion chromatogram) and XIC (extracted ion chromatogram).

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Q ExactiveTM is a Quadrupole-OrbitrapTM mass spectrometer. The Orbitrap is an ion trap mass analyser that is comprised of two outer electrodes and a central electrode, which functions as both an analyser and detector (Hu et al., 2005). The injected ions that enter the Orbitrap are trapped and squeezed by increasing electric field, making the ions oscillate around the axial and in between of the outer electrodes. Different ions oscillate at different frequencies and separate from one another. The measurements of the oscillation frequencies caused by ions reaching on the outer electrodes are converted into the mass spectra of the ions by image current detection. The Orbitrap mass analyser is actually a Fourier Transform mass analyser analogue of FT-ion cyclotron resonance (ICR) technology, with smaller instrument size and easier operation. Orbitrap mass spectrometer is able to identify, quantify and confirm more compounds rapidly and with more confidence. The nanometre-range electrodes, high voltage supply and wider range of mass detection provides better sensitivity, selectivity, resolution, and accuracy.

There are two solvents used for HPLC-MS/MS. Solvent A is 0.1% (v/v) formic acid (FA) and Solvent B is 0.1% (v/v) FA +80% (v/v) acetonitrile (ACN). HPLC solvents are prepared by core facility and replaced monthly. These solvents are sonicated for 10 min in a water bath sonicator to minimise the likelihood of introducing bubbles into the nanoHPLC system.

All proteolytic digested samples were separated by nano-liquid chromatography (nano-LC) using Ultimate 3000 HPLC and autosampler system (Dionex). A total of 10 μL of reconstituted peptide solution were loaded onto AcclaimTM PepMapTM C18 micro-column (Thermo Fisher) 15 μL/min. Peptides were eluted with a linear gradient of H2O: ACN (98:2, 0.1 % FA) to H2O:

ACN (64:36, 0.1 % FA) at 250 nL/min over 60 min with a nanoflow of 0.3 μL/min. Positive ions were generated by electrospray and captured by detector and analyser.

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A survey scan m/z 350-1800 was acquired in the Orbitrap operated in data dependent mode (Resolution = 60,000 at m/z 400, with automatic gain control target of 1,000,000 ions counts). Up to the 20 most abundant ions (>5,000 ion counts) with charges over +2 were sequentially isolated and fragmented by collision induced dissociation (CID). M/z ratios selected for MS/MS were dynamically excluded for 30 seconds.

Proteome Discoverer 2.2 (Thermo Fischer Scientific) and Mascot (Matrix Science) were used to collect and analyse MS data. The result data were converted to peak list files to query at Mascot search engine for MS/MS ions match in the SwissProt Database (uniprot_SwissProt_Human_2018-04.fasta) with an FDR (false discovery rate) <1%. The parameters used to search for protein were allowed up to two missed cleavages for trypsin; one fixed modification (carbamidomethyl of Cys); one variable modifications for oxidation of Met; mass tolerance of 10 ppm for the precursor ion and mass tolerance of 0.8 Da for the fragment ion. The search results included a list of identified peptides for further analysis. Scaffold 4 (Proteome Software) was used to combine and compare search results to identify biological relevance, display spectrum details and counts and create comprehensive lists of target proteins classified by their molecular function or involvement in biological processes.