DIA and data-dependent acquisition (DDA) MS analysis were used as previously described with some modification (25 (link)). Briefly, an iRT-kit (Biognosys, Schlieren, Switzerland) was employed in the peptide sample at a 1:10 ratio to correct the retention time. DDA- and DIA-MS analyses were performed with an Orbitrap Fusion Lumos mass spectrometer (Thermo, USA) equipped with an EASY-nLC 1000 (Thermo, USA). The peptides were separated on Omics high-resolution series monolithic capillary high-pressure liquid chromatography (HPLC) columns (100 μM × 50 cm; Kyoto Monotche) with a column temperature of 50°C. The MS parameters were performed as described previously (25 (link)). Raw DDA data sets were used to search against the UniProt E. coli K-12 database (4,356 entries) in the Sequest HT (Proteome Discoverer v2.2) local server and Biognosys Spectronaut software Pulsar (25 (link)). The DIA search parameters were also performed as described previously (25 (link)).
Irt kit
Manufactured by Biognosys
Sourced in Switzerland
The IRT kit is a laboratory equipment product developed by Biognosys. It is a tool designed for the quantification of proteins in complex biological samples using targeted mass spectrometry.
Automatically generated - may contain errors
Lab products found in correlation
Spectronaut pulsar x tm 12, by Biognosys (4 mentions)
Orbitrap fusion lumos mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Orbitrap q exactive hf x mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Easy nlctm 1200 uhplc system, by Thermo Fisher Scientific (3 mentions)
Spectronaut pulsar x, by Biognosys (3 mentions)
C18 macrospin column, by Nest Group (3 mentions)
Easy nlc 1000, by Thermo Fisher Scientific (2 mentions)
Spectronaut, by Biognosys (2 mentions)
Pierce hela protein digest standard, by Thermo Fisher Scientific (2 mentions)
Orbitrap fusion mass spectrometer, by Thermo Fisher Scientific (2 mentions)
U3000 uhplc system, by Thermo Fisher Scientific (2 mentions)
C18 ziptip, by Merck Group (2 mentions)
Spectronaut software, by Biognosys (2 mentions)
Bca protein assay kit, by Bio-Rad (2 mentions)
Spectronaut 15, by Biognosys (1 mentions)
Q exactive hf, by Thermo Fisher Scientific (1 mentions)
Easy nlc 1200 uhplc system, by Thermo Fisher Scientific (1 mentions)
Fusion lumos, by Thermo Fisher Scientific (1 mentions)
Easy nlc 1200 high, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Serva Electrophoresis (1 mentions)
49 protocols using irt kit
1
Quantitative Proteomics of E. coli
2
DIA Mass Spectrometry of Serum Peptides
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Each serum sample peptide was reconstituted in 0.1% FA, mixed with 0.2 µL standard peptides (iRT kit, Biognosys), and injected into the EASY‐nLCTM 1200 UHPLC system coupled with an Orbitrap Q‐Exactive HF‐X mass spectrometer (Thermo Fisher Scientific) operating in DIA mode. The liquid conditions were identical to those used in the DDA model. For DIA acquisition, the MS1 resolution was set to ×60,000 (at 200 m/z) and the MS2 resolution was set to ×30,000 (at 200 m/z). The m/z range was 350–1500 and was separated into 30 acquisition windows (Table S1 ). The full‐scan AGC target was set to 3 × 10 (Scott et al., 2004 (link)) with an injection time of 50 ms. The DIA settings included an NCE of 27%, a target value of 1 × 10 (Scott et al., 2004 (link)), and an automatic maximum injection time to allow the MS to operate continuously in parallel ion filling and detection mode.
3
DIA Proteomics Analysis of Organoids
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DIA proteomic analyses were performed as previously described (Muntel et al., 2019 (link)). Briefly, 10 organoids at D34 were collected as one group, and three samples were set for each group. The DIA was subcontracted to Shanghai Omicsolution Co., Ltd. (Shanghai, China). Six samples of RP and control groups were processed individually by DIA to assess their proteome differences. MS1 and MS2 data were all acquired by random order. The iRT Kit (Ki3002, Biognosys AG) was used to calibrate the retention time of the extracted peptide peaks. Statistical assessment of the DIA dataset, including data normalization and relative protein quantification, was performed using Spectronaut 15 (Biognosys AG). Uniprot (https://www.uniprot.org ) was used for retrieval of interacting genes and proteins and converted UniprotKB.ID of proteins into gene names. After DEGseq analysis, differently expressed proteins were filtered if the |fold change| ≥1.2 and Q-value <0.05.
4
LC-MS/MS Peptide Identification Protocol
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LC-MS/MS analysis was performed using an EASY-nLC 1200 UHPLC system and Q-Exactive HF (Thermo Scientific, Rockwell, IL, USA). All peptides dissolved in 0.1% FA were loaded onto the trap column. Subsequently, the eluent was transferred to a reversed-phase analytical column (75 μm × 500 mm, 2 μm, MONOTECH). The elution gradient was 5-30% buffer B (flow rate = 300 nl/min; 0.1% FA in 99.9% acetonitrile) over 120 min. An iRT kit (Biognosys AG, Schlieren, Switzerland) was used for retention time alignments in all samples. The parameters of MS were set as follows: the full scan was achieved by a resolution of 120,000 and in the range of 400-1,200 m/z; the cycle time was set at 3 s; the AGC was 3e6; the injection time was under 100 ms; charge state screening was performed by precursors with a +2 to +6 charge state; and the dynamic exclusion duration was 10 s. According to the precursor m/z distribution of the pooled sample, the number of precursor ions in each isolation window was equalized.
5
Quantitative Proteomics of E. coli
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DIA and data-dependent acquisition (DDA) MS analysis were used as previously described with some modification (25 (link)). Briefly, an iRT-kit (Biognosys, Schlieren, Switzerland) was employed in the peptide sample at a 1:10 ratio to correct the retention time. DDA- and DIA-MS analyses were performed with an Orbitrap Fusion Lumos mass spectrometer (Thermo, USA) equipped with an EASY-nLC 1000 (Thermo, USA). The peptides were separated on Omics high-resolution series monolithic capillary high-pressure liquid chromatography (HPLC) columns (100 μM × 50 cm; Kyoto Monotche) with a column temperature of 50°C. The MS parameters were performed as described previously (25 (link)). Raw DDA data sets were used to search against the UniProt E. coli K-12 database (4,356 entries) in the Sequest HT (Proteome Discoverer v2.2) local server and Biognosys Spectronaut software Pulsar (25 (link)). The DIA search parameters were also performed as described previously (25 (link)).
6
Constructing a DDA Spectral Library
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To construct the DDA library, the FASTA sequence database was searched using Spectronaut Pulsar X (version14.4, Biognosys AG, USA) after FASTA database download from the UniProt website (http://www.uniprot.org ) with the iRT peptides sequence added (>Biognosys|iRTKit|Sequence_fusionLGGNEQVTRYILAGVENSKGTFIIDPGGVIRGTFIIDPAAVIRGAGSSEPVTGLDAKTPVISGGPYEYRVEATFGVDESNAKTPVITGAPYEYRDGLDAASYYAPVRADVTPADFSEWSKLFLQFGAQGSPFLK). The parameters were set as follows: enzyme set to trypsin, max missed cleavage set to 2, fixed modification set to carbamidomethyl (C), dynamic modification set to oxidation(M) and acetyl (Protein N-term). All protein identification results were evaluated then significant results were selected based on 99% confidence, as determined using false discovery rate (FDR =N(decoy)∗2/(N(decoy)+ N(target))) ≤1%. The spectral library was constructed by importing original raw spectral files and search results into Spectronaut Pulsar X (version14.4, Biognosys AG, USA). DIA data were analyzed by searching the abovementioned constructed spectral library. Main software parameters were set as follows: retention time prediction type is dynamic iRT, interference on MS2 level correction is enabled, and cross run normalization is enabled. All results were filtered based on the P value cutoff of .01 (equivalent to FDR <1%).
7
Peptide Enrichment Using SCX Solid-Phase Extraction
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Trypsin digested proteins were resuspended in 2% ACN in 0.1% TFA and subjected to solid-phase extraction using a SCX membrane disk inserted into a StageTip [33 (link)]. Specifically, SCX material was activated using 100% ACN and conditioned with 5% ammonium hydroxide in 80% ACN. SCX membrane equilibration, sample loading, and two washes utilized 2% ACN in 0.1% TFA. Finally, the peptides were eluted using 5% ammonium hydroxide in 80% ACN. Samples were then dried under vacuum and peptides were resuspended using a solution containing 11 standard peptides (iRT Kit from Biognosys) made up in 2% ACN in 0.1% FA and submitted for mass spectrometry analysis.
8
Detailed LC-MS/MS Workflow for Peptide Analysis
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LC-MS/MS data acquisition was performed on a Fusion Lumos mass spectrometer (Thermo Scientific, Germany) coupled with an EASY-nLC 1200 high-performance liquid chromatography system (Thermo Scientific, Germany). For DDA-MS and DIA-MS modes, the same LC settings were used for retention time stability. The digested peptides were dissolved in 0.1% formic acid and loaded onto a trap column (75 µm × 2 cm, 3 µm, C18, 100 A˚), and the eluent was transferred to a reversed-phase analysis column (50 µm × 250 mm, 2 µm, C18, 100 A˚) with an elution gradient of 5-30% phase B (79.9% acetonitrile, 0.1% formic acid, flow rate of 0.3 μL/min) for 90 min. To enable fully automated and sensitive signal processing, the calibration kit (iRT kit, Biognosys, Switzerland) was spiked at a concentration of 1:20 v/v in all samples。 For the generation of the spectral library, 10 peptide fractions were analyzed in DDA-MS mode.
The parameters were set as follows. The full scan was acquired from 350 to 1 550 m/z at 60 000, and the cycle time was set to 3 s (top speed mode). The auto gain control (AGC) was set to 1e6, and the maximum injection time was set to 50 ms. The MS/MS scans were acquired in the Orbitrap at a resolution of 15000 with an isolation window of 2 Da and collision energy at 32% (HCD). The AGC target was set to 5e4, and the maximum injection time was 30 ms.
The parameters were set as follows. The full scan was acquired from 350 to 1 550 m/z at 60 000, and the cycle time was set to 3 s (top speed mode). The auto gain control (AGC) was set to 1e6, and the maximum injection time was set to 50 ms. The MS/MS scans were acquired in the Orbitrap at a resolution of 15000 with an isolation window of 2 Da and collision energy at 32% (HCD). The AGC target was set to 5e4, and the maximum injection time was 30 ms.
9
MaxQuant-Based Quantitative Proteomics Workflow
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MaxQuant software (1.5.3.17) was used to search the FASTA sequence database. The database was downloaded from http://www.uniprot.org , 20 September 2022. The iRT peptide sequence was as follows:
Biognosys|iRT-Kit|Sequence_fusionLGGNEQVTRYILAGVENSKGTFIIDPGGVIRGTFIIDPAAVIRGAGSSEPVTGLDAKTPVISGGPYEYRVEATFGVDESNAKTPVITGAPYEYRDGLDAASYYAPVRADVTPADFSEWSKLFLQFGAQGSPFLK.
The MS search parameters were as follows: cutting enzyme, trypsin; maximum missed cleavages, 2; fixed modification, carbamidomethyl (C); dynamic modifications, oxidation (M) and acetyl (Protein N-term). The reported data were based on 99% confidence for protein identification by false discovery rate (≤1%).
The spectral library was constructed by importing the original raw files, and DDA search results were imported into Spectronaut Pulsar XTM_12.0.20491.4 (Biognosys AG, Schlieren, Switzerland). Spectronaut was used to analyse the DIA data with the above constructed spectral library. The main software parameters were set as follows: retention time prediction type, dynamic iRT; interference on MS2 level correction, enabled; cross run normalisation, enabled. All of the results were filtered based on a Q value cut-off of 0.01 (equivalent to FDR < 1%).
Biognosys|iRT-Kit|Sequence_fusionLGGNEQVTRYILAGVENSKGTFIIDPGGVIRGTFIIDPAAVIRGAGSSEPVTGLDAKTPVISGGPYEYRVEATFGVDESNAKTPVITGAPYEYRDGLDAASYYAPVRADVTPADFSEWSKLFLQFGAQGSPFLK.
The MS search parameters were as follows: cutting enzyme, trypsin; maximum missed cleavages, 2; fixed modification, carbamidomethyl (C); dynamic modifications, oxidation (M) and acetyl (Protein N-term). The reported data were based on 99% confidence for protein identification by false discovery rate (≤1%).
The spectral library was constructed by importing the original raw files, and DDA search results were imported into Spectronaut Pulsar XTM_12.0.20491.4 (Biognosys AG, Schlieren, Switzerland). Spectronaut was used to analyse the DIA data with the above constructed spectral library. The main software parameters were set as follows: retention time prediction type, dynamic iRT; interference on MS2 level correction, enabled; cross run normalisation, enabled. All of the results were filtered based on a Q value cut-off of 0.01 (equivalent to FDR < 1%).
10
Quantitative E. coli Proteome Analysis
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Peptides were dissolved in 0.1% formic acid and diluted to 0.5 μg/μL. To build the precursor ion library, 1 μL of each sample was taken to prepare a mixed sample. IRT-standard provided by the iRT-Kit (Biognosys, Schlieren, Switzerland) at 1/10 by volume was added to all the samples. The Next, we used the following command to convert the DIA original file to htrm format: HTRMS converter provided by Spectronaut. Finally, the DIA htrm file, the DDA original file, the DDA pdResult file, and the customised Uniprot-E. coli K12 database + iRT standard peptide FASTA file were loaded into Spectronaut, and processed through the BGS factory settings. Basically, the default parameters were used, except the enzyme Modify from trypsin/P to trypsin and add acetylation of K. Protein was inferred by software, standard q value of 0.01 was used to obtain the quantitative information at protein level, which was used for subsequent analysis.
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