Materials and methods for proteomics analysis

M. pectinilyticus was grown using four different types of substrates: 0.5 % (w/v) D-fructose, 0.2 % (w/v) apple pectin, 0.2 % (w/v) citrus pectin, and 0.2 % (w/v) kiwifruit pectin. For each substrate, 5 × 50 ml of mineral medium containing 5 % (v/v) clarified rumen fluid was used as the base medium. Cultures were incubated at 37℃ with a constant shaking until stationary phase was reached. In order to enable the detection of the highest possible number of proteins potentially related to pectin metabolism, proteins were extracted from M. pectinilyticus cultures growing in a stationary phase in which the highest level of protein secretion is believed to take place (Antelmann et al., 2001; Voigt et al., 2006; Evans et al., 2007). Proteins containing an N-terminal signal peptide have also been reported to become expressed and secreted into the growth medium during the stationary phase since these proteins lack cell wall-retention domains such as SLH modules which show strong affinity for peptidoglycan cell wall (Antelmann et al., 2001; Rincon et al., 2005). Fully grown cultures were combined (250 ml each) and centrifuged at 12,000 g for 10 min. Cell pellets were stored frozen at - 80℃. The supernatant was filter-sterilized through 0.22 µm filters (Merck Millipore) and then concentrated by ultrafiltration using 10 kDa Amicon Ultra filters by centrifuging at 4,000 rpm for 20 min (Millipore Analytical). Supernatant proteins were further concentrated using 50 kDa Amicon Ultra filters by centrifuging at 4,000 rpm for 20 min (Millipore Analytical). Pectinase assay and Qubit™ protein assay was carried out for both > 50 kDa and < 50 kDa supernatant fractions to ensure a negligible amount of enzyme loss occurred during ultrafiltration process. Frozen cell pellets and > 50 kDa supernatant samples were sent to Auckland Science Analytical Services (University of Auckland) for quantitative proteomics analysis.

2.4.2 Pectinase assay

2.4.2.1 Imidazole-HCl buffer

Imidazole-HCl buffer (pH 7.2) contained 50 mM imidazole, 20 mM HCl, 1.5 g/L sodium azide, 10 mM CaCl2, 10 mM DDT, and 0.5% (w/v) citrus pectin.

2.4.2.2 DNSA reagent

DNSA solution contained 1% (w/v) 3,5-dinitrosalicylic acid, 0.05 % (w/v) sodium sulfite, and 1 % (w/v) sodium hydroxide. Solution was stored in dark overnight before use.

2.4.2.3 Pectinase assay method

Colorimetric assay was performed using DNSA assay method described by Kashyap et al., (2000), developed by Miller (1959), and modified by Aguillar and Huitron (1990) using galacturonic acid standard. 100 μl of concentrated supernatant proteins (> 50 kDa) and the filtrates (< 50 kDa) were added to 100 μl of imidazole-HCl assay buffer. Mixture was incubated at 36℃ for 3 hr under constant shaking. 400 μl of DNSA reagent were added and the mixture was boiled for 15 min to develop the color. Mixture was diluted with 4.4 ml of deionized water. Absorbance was measured at 530 nm using Jenway 6100 Spectrophotometer. Enzyme activity was calculated by defining one unit of enzyme as the amount of enzyme which catalyzes the formation of 1 μmol of reducing sugar per min at a fixed pH. Enzyme activity (U) per μg of protein was calculated.

2.4.3 iTRAQ quantitative proteomics 2.4.3.1 iTRAQ sample preparation

Three independently produced biological replicates of 4-plex iTRAQ were used to analyse the proteome profiles of whole-cell lysates. Due to the limited amount of resources allocated to this part of the study, supernatant proteins were analysed without biological or technical replicates. Cellular proteins were extracted by sonication of cell pellets in lysis buffer (50 mM ammonium bicarbonate containing 7M urea, 2M thiourea and 10 mM DTT). The total protein content of each sample was assayed using the EZQ Protein Quantitation Kit (Life Technologies). Aliquots containing 25µg of protein were taken for each sample and volumes adjusted to 100ul. Samples were reduced by

incubation at 56°C for 30 minutes in the presence of 10 mM DTT. Samples were cooled and alkylated with 50mM iodoacetamide (GE Healthcare) in the dark at RT for 30 minutes, and digested with 1µg of sequencing grade trypsin (Promega). Digests were acidified with formic acid, desalted on Oasis HLB SPE cartridges (Waters), and dried down in a vacuum centrifuge. Samples were reconstituted with 30ul of 0.5M TEAB (Sigma) and labelled with 4-plex iTRAQ labels (Sciex) according to the manufacturer’s supplied protocol. Labelling reactions were concentrated in a vacuum centrifuge and resuspended in 0.1% formic acid. Pools were prepared using equal amounts of each sample, and desalted on Oasis HLB cartridges as above.

2.4.3.2 LC-MS/MS and database search

Samples were injected onto a 0.3 × 10 mm trap column packed with Reprosil C18 media and desalted for 5 min at 2 µl/min before being separated on a 0.075 × 200 mm picofrit column (New Objective) packed in-house with Reprosil C18 media. The following gradient was formed at 300 nl/min using a NanoLC 400 UPLC system (Eksigent): 0 min 10 %B; 50 min, 35 %B; 52 min, 90 %B; 55 min, 90 %B; 56 min, 10 %B; 60 min, 10 %B, where A was 0.1 % formic acid in water and B was 0.1% formic acid in acetonitrile. The picofrit spray was directed into a TripleTOF 6600 Quadrupole-Time-of-Flight mass spectrometer (Sciex) scanning from 350-1200 m/z for 250 ms, followed by 40 ms MS/MS scans on the 40 most abundant multiply-charged peptides (m/z 60-1200) for a total cycle time of ~2

seconds. The mass spectrometer and HPLC system were under the control of the Analyst TF 1.7 software package (Sciex). The resulting data from each pool were searched against a database containing the UniProt sequences for pentapetalae from August 2015 (1,895,602 entries) appended to a custom database of M. pectinilyticus entries including common contaminant sequences (2418 entries) using ProteinPilot version 5.0 (Sciex). Raw iTRAQ peak area data were processed through a combination of Paragon™ and Pro Group™ algorithms to reduce the protein inference ambiguities coming from protein modifications and to bundle peptides into winner protein groups. False Discovery Rate analysis was enabled. Search parameters were as follows: Sample Type, iTRAQ 4- plex (Peptide Labelled); Search Effort, Thorough; Cys Alkylation, Iodoacetamide; Digestion, Trypsin: FDR analysis, Yes. Using the peak height of reporter ions as a proxy for marker mass abundance,

peptide ratios across 114 (apple), 115 (citrus), 116 (fructose), and 117 (kiwifruit) samples were determined in log space using 116 values as the baseline denominators. After performing bias correction by applying a correction factor of < 20 % across the samples, an average ratio was

calculated for each protein. P-values were calculated to assess the possibility of random distribution of peptide ratios contributing to protein inference. Peptide and Protein summary files were exported in Excel format for further statistical analyses.

2.4.3.3 Statistics

While the direction of changes in differential expression are not usually affected, iTRAQ experiments rarely reported fold-change ratios exceeding 2 (Pascovici et al., 2015). Therefore, the typically used 2- or 0.5-cutoffs to estimate up- or down-regulation of protein production are often considered too strict, and were readjusted with less stringent thresholds of 1.2- and 0.83-folds in this study (Pascovici et al., 2015). The large proportion of identified proteins represented by a single peptide is another issue causing a significant amount of data exclusion from the total proteome coverage in iTRAQ datasets (Evans et al., 2012). From the acquired dataset, it was noted that proteins represented by one or two unique peptides often suffer from a high statistical variability. Because reliability of fold- changes ratios are often directly proportional to the number of peptides contributing towards the ratio calculation, only those proteins represented by ≥ 3 unique peptides with > 95 % confidence intervals were included for further analysis in this study (Pascovici et al., 2015). To ensure the statistical significance of the dataset, unused protein score of > 2, and p-value of < 0.05 inferred from using ProteinPilot software (version 5.0) were coupled with the quantification data to remove unreliable peptide identification results. In ProteinPilot, unused scores reflect the amount of total and unique peptide evidence which identifies with the protein hit. While a protein with an unused score of 0 is considered unreliable as it suggests that no unique peptide was identified for this particular protein, an unused score of > 2 gives a solid metric for deciding if a protein has been confidently identified. In order to inspect the presence of potential peptide outliers, Q-Q plots were constructed for each protein by plotting the logarithm of peptide ratios against the expected normal distribution. The qqmaths function in R package lattice was used to produce the graphs.

2.5 Materials and methods for human carriage study