33 heavy isotope labeled neo-antigenic peptides flanking gene mutation hotspots on cancer driver genes (including K-Ras, EGFR, TP53, CTNNB1, and IDH2) were predicted by NetMHC 4.0 [20 (link)] and subsequently synthesized as AQUA™ Peptides by Sigma-Aldrich (Supplementary Table S2 ). Optimization of collision energies and fractionation were performed as previously described [19 (link)]. A list of the SRM transitions and fraction group IDs for all 33 peptides are shown in Supplementary Table S2 online. All transition parameters were manually examined and curated to exclude ions with excessive noise due to co-elution with impurities. Copy number of each detectable neo-antigenic peptide was calculated using heavy isotope-labeled peptides as previously described [21 (link)].
Aqua peptide
Manufactured by Merck Group
Sourced in United States
AQUA Peptides are a line of synthetic peptides produced by Merck Group for use in analytical laboratories. These peptides are designed to serve as internal standards for quantitative mass spectrometry analysis of proteins and peptides.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (1 mentions)
Bca protein quantification kit, by Thermo Fisher Scientific (1 mentions)
Ms grade trypsin protease, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor tablet, by Roche (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Ammonium bicarbonate, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Mem per plus kit, by Thermo Fisher Scientific (1 mentions)
Iq sybr supermix, by Bio-Rad (1 mentions)
Mascot v2, by Matrix Science (1 mentions)
Mascot 2, by Matrix Science (1 mentions)
Tsk gel g2000 swxl column, by Tosoh (1 mentions)
Qtrap 6500 mass spectrometer, by AB Sciex (1 mentions)
8 protocols using aqua peptide
1
Targeted Multiplexed Quantification of Cancer Neoantigens
2
Optimized Protein and RNA Extraction Workflow
3
Mass Spectrometry-based Proteomics Analysis
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Peak lists were created using an in-house Perl script based on the recorded product ion mass spectra. Peptides and proteins were identified with Mascot v2.4 (Matrix Science, London) against the UniProt/Swiss-Prot database (downloaded 2013/06, subset C. elegans, 3428 protein entries) in error-tolerant mode [25 (link)] or in an in-house protein database (in which all Gly residues arising from the decoding of the GGG codons were altered to Leu or Ser). A precursor mass tolerance of 3 ppm and a fragment ion mass tolerance of 0.8 Da were used with strict enzyme specificity, allowing for up to two missed cleavages [45 (link)]. The carbamidomethylation of cysteine was set as a fixed modification, and methionine oxidations were allowed as variable modifications. Peptides were rejected if the Mascot score was below the 95% confidence limit based on the “identity” score for each peptide. Some of the identified peptides were further validated with a targeted proteomics approach using synthesized peptides (AQUA Peptides; Sigma-Aldrich, St. Louis, MO, USA) that contained stable-isotope-labeled amino acids (see S2 and S3 Tables).
4
Absolute Quantification of Peptides
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The abundance of a target peptide was represented by the total area under the curve (AUC) of all its transitions normalized to the total AUC of all transitions from the most nearby (sharing a similar hydrophobicity) heavy isotope-labeled peptide from MyPro-SRM Internal Control Mixture (MyOmicsDx, Inc) spiked in before the SRM analysis. Absolute quantification of each protein is performed through applying AQUA Peptides purchased from Sigma-Aldrich. A summary of all quantification results were shown in S8 Table .
5
Synthetic β-PRVB peptide epitopes
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Selected B-cell peptide epitopes from β-PRVB were chemically synthetized (Track Peptide Libraries, JPT Peptide Technologies GmbH, Berlin, Germany) (Table 3). The lyophilized peptides were individually reconstituted with 1x PBS and stored at -80°C.
Additionally, as negative control peptides were used synthetic peptides with the same length (7 residues) but with a shuffled amino acid sequence (ELLINVK) belonging to the alcohol dehydrogenase protein of Sacharomyces cerevisiae and a purity of 91.2% (AQUA Peptides, Sigma-Aldrich, S.L., Madrid, Spain).
Additionally, as negative control peptides were used synthetic peptides with the same length (7 residues) but with a shuffled amino acid sequence (ELLINVK) belonging to the alcohol dehydrogenase protein of Sacharomyces cerevisiae and a purity of 91.2% (AQUA Peptides, Sigma-Aldrich, S.L., Madrid, Spain).
6
Protein Characterization by SE-HPLC and LC-MS/MS
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The MW distribution was determined according to Wang et al. [17 (link)] using size exclusion (SE)-HPLC with a TSK gel G2000 SWXL column (7.8 × 300 mm, TOSOH, Tokyo, Japan) for analysis. Peptide sequences of WPH were identified using liquid chromatography–tandem mass spectrometry (LC-MS/MS) and searched in the MASCOT 2.4 search engine (Matrix Science, Boston, MA, USA) based on UniProt Knowledgebase (UniProtKB) database. The result was quantified by calibration curve using AQUA peptide (Sigma Aldrich, Shanghai, China) as a standard.
7
Peptide Quantification by Stable Isotope Labeling
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Multiple point calibration was used where a series of standard ‘light’ peptides with known concentrations together with fixed amounts of stable isotope-labelled ‘heavy’ peptides (100 fmol/μl) were used to generate calibration curves (Fig. 1 ). The curves were expressed as ratios of light/heavy peak area (y-axis) versus concentration of light peptide (x-axis) for each of the 14 peptides selected. A commercially available stable isotope-labelled peptide standard (AQUA peptide, Sigma, USA) was used for absolute quantification of proteins. This heavy surrogate of each of the peptide standards is added at a constant level to all samples and standards and is used for correction of sample losses during workup and normalisation across runs, allowing accurate quantification of the target protein in samples. Peptides labelled with a stable isotope (13C and 15N) are chemically identical to their native counterparts and have identical chromatographic behaviour but can be distinguished from the calibration standards based on a small specific mass difference.
8
Quantifying P-gp and Villin in Human Intestine
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The absolute expression levels of P-gp and villin at the outer plasma membrane of human intestinal samples (jejunum, n = 4) were determined as previously described (Bosgra et al., 2014; van de Steeg et al., 2013) . Samples were processed in duplicate (except for one sample, which was processed in mono due to insufficient amounts of tissue), and approximately 350 mg human intestinal tissue was used for plasma membrane isolations per run. After trypsin digestion, the samples were analyzed by a UPLC coupled to a 6500 QTrap mass spectrometer (AB Sciex). The peptide sequence was chosen according to the in silico peptide criteria defined by Kamiie et al. (2008) and is exclusively present in the target protein of interest (i.e. P-gp or villin). For each peptide, three transitions (Q3-1, Q3-2, and Q3-3) were used for quantitation and confirmation, listed in Table 2. A peptide labeled with 15 N and 13 C (AQUA peptide) was synthesized (Sigma Aldrich, Steinheim DE) and used as an internal standard for quantification (Table 2). Peak identification and quantification was performed using Masslynx software version 4.1.
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