Novel and cost‐effective 6‐plex isobaric tagging reagent, DiART, is effective for identification and relative quantification of complex protein mixtures using PQD fragmentation

Abstract

Deuterium isobaric Amine Reactive Tag (DiART) reagents facilitate relative quantification during proteomic analysis in a functionally similar manner to commercially available isobaric tag for relative and absolute quantitation (iTRAQ) and tandem mass tag (TMT) reagents. In contrast to iTRAQ and TMT, DiART reagents incorporate deuterium isotopes which significantly reduce the number of required synthesis steps and hence have potential to significantly reduce reagent production cost. We examined the capability of DiART for performing quantitative proteomic experiments using a linear ion-trap mass spectrometer with Pulsed Q Dissociation (PQD) fragmentation. Using a synthetic peptide tagged with DiART reagent, we observed a precise mass shift of 144.79 Da on the triply charged precursor ion, which shows complete derivatization of the N-terminus and ε-amino group of lysine. A DiART tagged tryptic digest of bovine serum albumin showed a sequence coverage of 57.99% which was very comparable to that showed by iTRAQ, 54.77%. Furthermore, a ten protein mixture tagged with DiART reagents and mixed in 1:1:1:1:1:1 exhibited < 15% error, whereas a linear trend (slope of 1.085) was observed when tagged proteins were mixed in the ratio 2:1:2:4:10:14 and plotted against theoretical ratios. Finally, when complex cell-wall protein mixtures from the model fungus A. nidulans were tagged with DiART reagents and mixed in different ratios, they exhibited similar trends. We conclude that DiART reagents are capable of performing quantitative proteomic experiments using PQD on a linear ion trap mass spectrometer.

Nano-liquid chromatography-mass spectrometry and recent applications in omics investigations

“…86 The analysis was performed by splitting each aorta extraction sample into three different analyses. The analyses were split to provide increased metabolic coverage using untargeted unlabeled analysis as well as isotopic labels for amine (DiART [87][88][89][90] ) and carbonyl (CILAT 91,92 ) analysis. All three sample regimes were separated using the same nLC column by simply changing separation conditions.…”

Methodologies and Perspectives of Proteomics Applied to Filamentous Fungi: From Sample Preparation to Secretome Analysis

“…By using a similar technical approach, secretome alteration of industrially relevant lignocellulose degrading fungi, such as Aspergillus niger wild type and mutant strains [ 46 ], Phanerochaete chrysosporium [ 91 , 92 ] and Trichoderma reesei [ 47 , 93 ], were dug into deeply, detecting a number of biotechnologically significant enzymes. Finally, a novel isobaric tag, named deuterium ((2)H) isobaric amine-reactive tag (DiART) [ 94 ], has been introduced for quantitative proteomics and used to study Aspergillus nidulans cell wall proteins [ 95 ].…”

Neuroproteomics — LC-MS Quantitative Approaches

“…[111,117] In a study comparing DiART and iTRAQ, the authors found that DiART leads to more intense reporter ions and consequently less ratio compression, however with the DiART approach, the common fragmentation method is not advisable due to easy reporter ion fragmentation [118]. DiART has also proven to be compatible and valuable for PTM analysis as quantitative phosphoproteomic studies [119].…”