A Sulfoxide‐Based Isobaric Labelling Reagent for Accurate Quantitative Mass Spectrometry

Abstract

Modern proteomics requires reagents for exact quantification of peptides in complex mixtures. Peptide labelling is most typically achieved with isobaric tags that consist of a balancer and a reporter part that separate in the gas phase. An ingenious distribution of stable isotopes provides multiple reagents with identical molecular weight but a different mass of the reporter groups, allowing relative quantification of multiple samples in one measurement. Here we report a new isobaric labelling reagent, where the balancer and the reporter are linked by a sulfoxide group, which, based on the sulfoxide pyrolysis, leads to easy and asymmetric cleavage at low fragmentation energy. The fragmentation of our new design is significantly improved, yielding more intense complementary ion signals, allowing complementary ion cluster analysis as well.

A Review on Quantitative Multiplexed Proteomics

“…Upon breakage, the isobaric tag produces low m / z reporter ions that contain different masses, depending on the conditions from which they originate, and can therefore be used for relative quantification (Figure B). Additionally, breakage of the isobaric tag leads to the formation of complement reporter ions, which contain the balancing part of the isobaric tag and the intact peptide . The balancing group of the isobaric tag also encodes the experimental conditions, and the complement reporter ions can therefore be similarly used for quantification (Figure B) .…”

“…F) Stadlmeier et al. developed the sulfoxide‐based tag, which was optimized for complement reporter ion formation due to fragmentation of the sulfoxide bond at lower energies . The two tertiary amines result in higher charge states of peptides after ionization and further facilitate fragmentation.…”

“…Motivated by the inefficient formation of the complement reporter ions (Figure B) from the commercial isobaric tags, Stadlmeier et al. developed a sulfoxide‐based tag, known as SO‐tag (Scheme F) . In this tag, the reporter and balancer groups are linked by a sulfoxide group.…”

“…In a proof of principle, we demonstrated the quantification of two different peptides in one spectrum . Particularly attractive might be the use of b or y ions that additionally have a broken isobaric tag, thereby forming complement fragment ions, which can also be used for quantification . Regardless, several hurdles have to be overcome to make this approach feasible.…”

Mass Spectrometry Advances and Perspectives for the Characterization of Emerging Adoptive Cell Therapies

“…On the other hand, designer mass tags are used to differently barcode peptides obtained from enzymatic digestion. Two designs are commercially available: tandem mass tags (TMTs) [60] and isobaric tags for relative and absolute quantification (iTRAQ) [61], and other designs, such as di-leucine (DiLeu) [62] or the sulfoxide-based isobaric reagents [63] are synthesized in house. In DIA, proteins are quantified via a label-free approach whereby the area-under-the-curve of the elution profile of select fragment ions is measured [30].…”