Free Radical Initiated Peptide Sequencing for Direct Site Localization of Sulfation and Phosphorylation with Negative Ion Mode Mass Spectrometry

Abstract

Tandem mass spectrometry (MS/MS) is the primary method for discovering, identifying, and localizing post-translational modifications (PTMs) in proteins. However, conventional positive ion mode collision induced dissociation (CID)-based MS/MS often fails to yield site-specific information for labile and acidic modifications due to low ionization efficiency in positive ion mode and/or preferential PTM loss. While a number of alternative methods have been developed to address this issue, most require specialized instrumentation or indirect detection. In this work, we present an amine-reactive TEMPO-based free radical initiated peptide sequencing (FRIPS) approach for negative ion mode analysis of phosphorylated and sulfated peptides. FRIPS-based fragmentation generates sequence informative ions for both phosphorylated and sulfated peptides with no significant PTM loss. Furthermore, FRIPS is compared to positive ion mode CID, electron transfer dissociation (ETD), as well as negative ion mode electron capture dissociation (niECD) and CID, both in terms of sequence coverage and fragmentation efficiency for phospho- and sulfo-peptides. Because FRIPS-based fragmentation has no particular instrumentation requirements and shows limited PTM loss, we propose this approach as a promising alternative to current techniques for analysis of labile and acidic PTMs.

Gas-Phase Hydrogen/Deuterium Scrambling in Negative-Ion Mode Tandem Mass Spectrometry

“…However, in contrast to nCID of this peptide, no imidazole C-2 hydrogen migration was observed. We recently calculated that TEMPO-assisted nFRIPS requires approximately 50 kcal mol −1 to promote homolytic cleavage and subsequent backbone dissociation [35]. Our current result implies: 1) that heteroatom-hydrogen scrambling requires less than 50 kcal mol −1 to proceed, and 2) that histidine C-2 and C β hydrogen migration likely requires more than 50 kcal mol −1 .…”

“…D 2 O (99.9 atom % D) was purchased from Sigma-Aldrich (St. Louis, MO). o-TEMPO-Bz-NHS was synthesized as described previously [35]. All other chemicals were obtained at HPLC grade from Thermo Fisher Scientific (Waltham, MA) and used without further purification.…”

“…Upon low level collisional activation, this radical precursor undergoes homolytic cleavage, generating a radical that propagates throughout the peptide to yield a neutral loss, or an a-x-, c-, or z-type ion (Scheme S1). TEMPO-assisted FRIPS was recently applied to peptide anions [35,42] and was shown to allow backbone cleavage without loss of highly labile posttranslational modifications, e.g., sulfation [35], suggesting potential applicability towards deuterated peptides. nFRIPS of P1 generated a near complete set of z-type ions (Figure S6).…”

“…Negative-ion mode MS/MS is gaining interest for proteomic analysis due to the importance of acidic post-translational modifications [30][31][32][33][34][35][36][37]. While the use of HDX in this polarity has been limited, applications towards carbohydrates and MALDI-generated peptide anions have been reported [38,39] with prevalent H/D scrambling observed in MALDI tandem time-offlight CID of peptide anions [39].…”

“…Here, we measure the extent of H/D scrambling in several negative-ion mode MS/MS techniques, including negative-ion mode CID (nCID), electron detachment dissociation (EDD) [40,41], negative-ion electron capture dissociation (niECD) [34], and negative-ion mode free radical induced peptide sequencing (nFRIPS) [35,42,43]. Observed scrambling behaviors lend insight into the energetics of these processes.…”