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Mode | Technique | Typical methods | Brief description | Quantification mode and benefits | Drawbacks | Example application to IBD research |
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Discovery | Two-dimensional electrophoresis [199] | 2DE, DIGE | Gel-based separation of proteins employing immobilized pH gradients and polyacrylamide gels. Protein spot patterns compared between gels for each sample to identify differences in protein (spot) abundance between samples. DIGE technology uses spectrally resolvable fluorescent dyes to label up to 3 samples with different dyes and separate them on the same gel, for intra-gel quantitation. | Relative quantification, a low-cost approach to protein separation and sample analysis. Applicable to analysis of proteoforms [39] | Protein identification requires an additional MS step | Samples: serum profiles of the early-stage vs. acute-stage CD in comparison to healthy controls. Key finding: data suggest C3c proteolytic isoform expression in Crohn’s disease could be disease-specific [188] |
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Discovery | Metabolic labelling | SILAC [200], Super-SILAC[201], and SILAMi [191] | In vivo protein labelling. Use of heavy, medium, and light isotopes allows discrimination from unlabeled (light) and thus relative quantification of peptides between samples | Relative quantification at MS level. Super-SILAC employs heavy reference standard enabling use of reference cell lines in patient studies | Not suitable for clinical material unless employing Super-SILAC for comparison to a common reference sample (cell line panel). Limited to comparison of 3 sample types (light, medium, and heavy SILAC labels) | Samples: paediatric control and biopsies of patients with CD and UC. Super-SILAC-based study. Key finding: identification of a panel of protein biomarkers to differentiate patients with CD from those with UC [177, 178] |
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Discovery | Chemical tagging [43] | iTRAQ, TMT [202–204] | In vitro labelling at the peptide level achieves simultaneous protein identification and quantification in multiplex format | Relative quantification at the MS2 level based on specific reporter ion intensities [203, 204]. Multiplex capability facilitates inclusion of replicates | Underestimation of fold change in relative protein amounts | Sample type: biopsy samples from UC patient groups differing in cancer risk and inflammatory status and healthy control. Key finding: acute inflammation resulted in reduced levels of intermediate filament proteins (keratins 8,18, and 19 and vimentin) compared to control and noninflamed mucosa as determined using iTRAQ analysis [172] |
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Discovery and targeted | Label-free methods (LFQ) [205, 206] | Used in Data Dependent acquisition and data-independent acquisition (MSE [207, 208], SWATH) [209, 210] approaches | Peptide intensity-based measurements, based on peak integration or spectral counting at MS level. | Relative quantification across multiple samples | Quantification achieved from independent sample runs. It is thus dependent on highly reproducible HPLC separations prior to MS. MSE and SWATH experiments enable retrospective data mining for specific peptides for relative quantification. | Sample type: colon tissue biopsies from normal, active, and inactive UC, inflammatory polyps, active and inactive CD. Key finding: three novel proteins, PRG2, LCP1, and PSME1, identified as candidate biomarkers signifying active CD [104] |
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Targeted | Targeted proteomic approaches [211] | Selected reaction monitoring (SRM) and related parallel reaction monitoring approach. When SRM is applied to investigate multiple product ions, this method is referred to as MRM | Quantification is based on detection and measurement of quantotypic peptides that represent the protein, based on a unique (proteotypic) amino acid sequence that is quantotypic, i.e., unique to the protein and stoichiometric to the amount of corresponding protein. | Relative quantification absolute quantification by reference to synthetic light and 13C/N15-labelled heavy peptides. Assays can be in multiplex format. | Cost of synthetic peptides and method optimisation | Sample type: plasma–low mass peptides. Key finding: peptides derived from secreted phosphoprotein 24 (SPP24) differentiates IBD from control. Active and quiescent disease can differentiate in UC and CD by secretogranin 1 and alpha-1-microglobulin [213] |
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