Protein bands for identification were excised and processed as described previously (Byrne et al., 2012 ) using both trypsin or chymotrypsin as the digestion enzymes. Proteolytic peptides (15 μL) were analysed as described previously (Colgrave et al., 2014 ) with chromatographic separation using a nano HPLC system (Shimadzu Scientific, Rydalmere, NSW, Australia) directly coupled to a 5600 TripleTOF MS (SCIEX, Foster City, CA). ProteinPilotTM 4.0 software (SCIEX) with the Paragon Algorithm (Shilov et al., 2007 ) was used for protein identification. Tandem mass spectrometry data were searched against in silico tryptic digests of Poaceae proteins of the Uniprot database (version 2014/07; 665 783 sequences). The search parameters were defined as iodoacetamide modified for cysteine alkylation and trypsin (or chymotrypsin) as the digestion enzyme. Modifications and cleavages were defined previously (Colgrave et al., 2014 ). The database search results were manually curated to yield the protein identifications using a 1% global false discovery rate (FDR) determined by the in‐built FDR tool within ProteinPilot software (Tang et al., 2008 ).
Nano hplc system
Manufactured by Shimadzu
Sourced in Australia, United States
The Nano HPLC system is a high-performance liquid chromatography instrument designed for the analysis of small sample volumes. It features a compact design and high-sensitivity detection capabilities, making it suitable for various applications in fields such as proteomics, metabolomics, and environmental analysis.
Automatically generated - may contain errors
Lab products found in correlation
Tripletof 5600 ms, by AB Sciex (2 mentions)
Tripletof 5600 ms system, by AB Sciex (1 mentions)
Proteinpilot 5, by AB Sciex (1 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Acclaim pepmap c18 reversed phase column, by Thermo Fisher Scientific (1 mentions)
Reversed phase c18 column, by Agela Technologies (1 mentions)
4 protocols using nano hplc system
1
Protein Identification by Mass Spectrometry
2
Nano-HPLC-TripleTOF 5600 MS Proteomic Analysis
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LC-MS/MS was performed using a TripleTOF 5600 MS System (AB Sciex, Foster City, CA, USA) coupled to a nano-HPLC system (Shimadzu). The peptides of each fraction from the SCX were separated by nano-HPLC on an in-house packed 12 cm × 75 μm Ultimate XB-C18 column (3 μm, 120 Å; Welch Materials Inc., Shanghai, China) at a flow rate of 300 nL/min. Each fraction was loaded in buffer C (5% ACN, 0.1% formic acid (FA)) and eluted with a linear 40-min gradient of 5%–45% buffer D (95% ACN, 0.1% FA). MS parameters were set as follows: electrospray voltage of 2.5 kV, positive ion data-dependent scan mode, full scan range of 350–1500 m/z, selection of the top 30 ions, and dynamic exclusion duration 18 s.
3
Gluten Proteome Identification Using Mass Spectrometry
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Gluten-enriched fractions (5 μL; corresponding to 5 μg extracted protein) were analyzed as described previously (Colgrave et al., 2014 (link)) with chromatographic separation using a nano HPLC system (Shimadzu Scientific, Rydalmere, Australia) directly coupled to a TripleTOF 5600 MS (SCIEX, Redwood City, CA, United States). ProteinPilotTM 5.0 software (SCIEX) with the Paragon Algorithm (Shilov et al., 2007 (link)) was used for protein identification. Tandem mass spectrometry data collected in this study was searched against the Poaceae subset of the Uniprot database (version 2018/08; 1,693,876 sequences). The search parameters were defined as iodoacetamide modified for cysteine alkylation and either trypsin or chymotrypsin as the digestion enzyme. Modifications and cleavages were defined previously (Colgrave et al., 2014 (link)). The database search results were manually curated to yield the protein identifications (Supplementary Tables S1 , S2 ) using a 1% global false discovery rate (FDR) determined by the in-built FDR tool within ProteinPilot software (Tang et al., 2008 (link)).
4
Desalted Peptide Separation and Analysis
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The desalted peptide mixture was delivered in duplicate onto a Acclaim PePmap C18-reversed phase column (75 μm × 2 cm, 3 μm, Thermo Scientific, California, USA) and separated with reversed phase C18 column (75 μm × 10 cm, 5 μm, Agela Technologies, Wilmington, Delaware, USA) mounted in a nano-HPLC system (SHIMADZU, Kyoto, Japan). Peptides were eluted using a gradient of 5–80% (v/v) acetonitrile in 0.1% formic acid (FA) over 45 min at a flow rate of 300 nL/min combined with a Q Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).
The eluates were directly entered Q-Exactive MS, setting in positive ion mode and data-dependent manner with full MS scan from 350 to 2000 m/z, full scan resolution at 70,000, MS/MS scan resolution at 17,500 with minimum signal threshold 1E + 5, isolation width at 2 Da. To evaluate the performance of this mass spectrometry on the iTRAQ labeled samples, two MS/MS acquisition modes, higher collision energy dissociation (HCD) was employed. And to optimize the MS/MS acquisition efficiency of HCD, normalized collision energy (NCE) was systemically examined 28, stepped 20%. Analysis was carried out with 3 technical replications.
The eluates were directly entered Q-Exactive MS, setting in positive ion mode and data-dependent manner with full MS scan from 350 to 2000 m/z, full scan resolution at 70,000, MS/MS scan resolution at 17,500 with minimum signal threshold 1E + 5, isolation width at 2 Da. To evaluate the performance of this mass spectrometry on the iTRAQ labeled samples, two MS/MS acquisition modes, higher collision energy dissociation (HCD) was employed. And to optimize the MS/MS acquisition efficiency of HCD, normalized collision energy (NCE) was systemically examined 28, stepped 20%. Analysis was carried out with 3 technical replications.
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