Protected Amine Labels: A Versatile Molecular Scaffold for Multiplexed Nominal Mass and Sub-Da Isotopologue Quantitative Proteomic Reagents

Abstract

We assemble a versatile molecular scaffold from simple building blocks to create binary and multiplexed stable isotope reagents for quantitative mass spectrometry. Termed Protected Amine Labels (PAL), these reagents offer multiple analytical figures of merit including, (i) robust targeting of peptide N-termini and lysyl side chains, (ii) optimal mass spectrometry ionization efficiency through regeneration of primary amines on labeled peptides, (iii) an amino acid-based mass tag that incorporates heavy isotopes of carbon, nitrogen, and oxygen to ensure matched physicochemical and MS/MS fragmentation behavior among labeled peptides, and (iv) a molecularly efficient architecture, in which the majority of hetero-atom centers can be used to synthesize a variety of nominal mass and sub-Da isotopologue stable isotope reagents. We demonstrate the performance of these reagents in well-established strategies whereby up to four channels of peptide isotopomers, each separated by 4 Da are quantified in MS-level scans with accuracies comparable to current commercial reagents. In addition we utilize the PAL scaffold to create isotopologue reagents in which labeled peptide analogs differ in mass based on the binding energy in carbon and nitrogen nuclei, thereby allowing quantification based on MS or MS/MS spectra. We demonstrate accurate quantification for reagents that support 6-plex labeling and propose extension of this scheme to 9-channels based on a similar PAL scaffold. Finally we provide exemplar data that extends the application of isotopologe-based quantification reagents to medium resolution, quadrupole time-of-flight mass spectrometers.

MEF2C Phosphorylation Is Required for Chemotherapy Resistance in Acute Myeloid Leukemia

“…The activity of MEF2C is known to be regulated by post-translational modifications, including acetylation, sumoylation, and phosphorylation, that can affect the recruitment of transcriptional co-repressors and co-activators (38–40). Analysis of existing phospho-signaling data revealed the presence of MEF2C S222 phosphorylation in human K562 leukemia cells and cytokine-stimulated hematopoietic progenitor cells (41,42). The specific association of MEF2C pS222 with failure of induction chemotherapy raises the possibility that MEF2C S222 phosphorylation promotes chemotherapy resistance in AML.…”

MEF2C phosphorylation is required for chemotherapy resistance in acute myeloid leukemia

“…The activity of MEF2C is known to be regulated by post-translational modifications, including acetylation, sumoylation, and phosphorylation, that can affect the recruitment of transcriptional co-repressors and co-activators (Kang et al, 2006;Ma et al, 2005;McKinsey et al, 2002). Analysis of existing phosphosignaling data revealed the presence of MEF2C S222 phosphorylation in human K562 leukemia cells and cytokine-stimulated hematopoietic progenitor cells (Ficarro et al, 2014;Zhou et al, 2013). The specific association of MEF2C pS222 with failure of induction chemotherapy raises the possibility that MEF2C S222 phosphorylation promotes chemotherapy resistance in AML.…”

Enhanced Multiplexing Technology for Proteomics

“…TMTs, TMTpro, and iTRAQ can multiplex 11, 18, and 8 samples, respectively, and also have unique nonisobaric versions called mTMT (93) and mTRAQ (94), which use the complementary reporter ion fragments for quantitation. Many isobaric tags have been modeled after amino acids because they are inexpensive and readily available for isotopic incorporation (61,62,95,96). Tags such as deuterium isobaric amine reactive tags (DiARTs), isobaric tags, and dimethyl leucine (DiLeu) (97,98) utilize leucine as a scaffold and are less costly compared to commercial reagents such as TMTs and iTRAQ (Table 1) (66,69,99).…”