Sciex 5600

Freeze-dried, ice-affinity purified material was sent to the Medical University of South Carolina (MUSC) and later to the College of Charleston (Charleston, SC), for liquid chromatography/mass spectrometry (LC/MS) analysis of tryptic peptides. Approximately 20 μg of total protein (as estimated from gel electrophoresis) was digested overnight at 37°C with trypsin gold (1:20; Promega) or GluC (1:10; Promega). Peptides were separated using two LC/MS platforms, an Eksigent nano-LC coupled to a SCIEX 5600 with a nanospray source and a Dionex Ultimate 3000 nano-LC coupled to an Orbitrap Fusion Lumos with a nanospray source at Hollings Marine Laboratory (Charleston, SC). Data were acquired from the Eksigent platform as described previously (39 (link)) and from the Dionex platform as described previously (40 (link)). Masses of the tryptic peptides were compared with predicted tryptic peptides in the Nostoc contig file translated in all six frames using MASCOT and MS-GF+ search engines. Searches included standard variable modifications and carbamidomethyl-fixed modifications. The search parameter Enzyme was set to trypsin or semitrypsin for data from trypsin digests or GluC(DE) for GluC endopeptidase digests. Error-tolerant searches were conducted to look for modified peptides not otherwise included.

Protein digestion, labelling and mass spectrometry was done as described in detail previously^{28 (link)}. 30 kDa size exclusion filters (Pall Corporation) were used in sample preparation. Samples were labelled with the isobaric iTRAQ tags as shown in Fig. 1 and pooled together for the mass spectrometry analysis. Labelled samples were analysed by Nano-RPLC- TripleTOF instrumentation using Eksigent ekspert™ 425 NanoLC coupled to high speed TripleTOF™ mass spectrometer (Sciex 5600+). A capillary RP-LC column (cHiPLC® ChromXP C18-CL, 3 µm particle size, 120 Å, 75 µm i.d × 15 cm; Eksigent, Concord, Canada) was used for LC separation of peptides.

Dimethyl labelled proteins were analysed by two mass spectrometers; the Sciex 5600 and the Thermo Scientific Q Exactive™. For full technical set up and method details see supplementary materials (section S10.4).

Dimethyl labelling of M. pneumoniae proteins was carried out as described previously^{28 (link),64 (link)}. 1 mg of M. pneumoniae protein was labelled in 40 mM formaldehyde (Ultrapure grade, Polysciences), 20 mM sodium cyanoborohydride, 100 mM Hepes (pH 6.7) for 4 hours at 37 °C. The reaction was quenched with 100 mM ammonium bicarbonate, precipitated in acetone:methanol (8:1), and digested with trypsin.
Peptides were analysed using both the Sciex 5600 and Thermo Scientific Q Exactive™ mass spectrometers. The methods, protocols, and parameters used have been described previously^{28 (link)}.