Discrimination Between Peptide O-Sulfo- and O-Phosphotyrosine Residues by Negative Ion Mode Electrospray Tandem Mass Spectrometry

Abstract

Unambiguous differentiation between isobaric sulfated and phosphorylated tyrosine residues (sTyr and pTyr) of proteins by mass spectrometry is challenging, even using high resolution mass spectrometers. Here we show that upon negative ion mode collision-induced dissociation (CID), pTyr- and sTyr-containing peptides exhibit entirely different modification-specific fragmentation patterns leading to a rapid discrimination between the isobaric covalent modifications using the tandem mass spectral data. This study reveals that the ratio between the relative abundances of [M-H-80](-) and [M-H-98](-) fragment ions in ion-trap CID and higher energy collision dissociation (HCD) spectra of singly deprotonated +80 Da Tyr-peptides can be used as a reliable indication of the Tyr modification group nature. For multiply deprotonated +80 Da Tyr-peptides, CID spectra of sTyr- and pTyr-containing sequences can be readily distinguished based on the presence/absence of the [M-nH-79]((n-1)-) and [M-nH-79-NL]((n-1)-) (n=2, 3) fragment ions (NL=neutral loss).

Direct Identification of Tyrosine Sulfation by using Ultraviolet Photodissociation Mass Spectrometry

“…Differentiating sulfotyrosine from phosphotyrosine is challenging based on the nearly isobaric nature of the modifications, with sulfation adding 79.956 Da and phosphorylation adding 79.966 Da. Other studies have approached the problem by exploiting differences in the neutral loss characteristics of phosphotyrosine- and sulfotyrosine-containing peptide anions upon CID [30]. Based on the UVPD fragmentation behavior of four deprotonated phosphotyrosine-containing peptides including TSTEPQpYQPGENL, Ac-DpYVPML-NH 2 , RRLIEDAEpYAARG, and Ac-IpYGEF-NH 2 , it appears that a similar method monitoring sulfo and phospho neutral losses can be used for UVPD.…”

Gas Phase Chemistry of Li+with Amides: the Observation of LiOH Loss in Mass Spectrometry

“…In the past few decades, continuous efforts have been devoted to the investigation of the dissociation reactions of charged compounds derived from protonation [4][5][6][7][8] or deprotonation [9][10][11][12][13]. On the other hand, the mass spectrometric fragmentations of metal cationized, especially lithiated biomolecules, have attracted much attention because of the desire to measure intrinsic metal ion affinity and identify the binding sites [14,15], and because the interesting dissociation pathways exhibited by these compounds are usually different from the protonated and deprotonated analogues [16,17].…”

Analysis of coagulation factor IX in bioreactor cell culture medium predicts yield and quality of the purified product

“…4B, inset). Other analytical workflows, including higher resolution positive ion mode or negative ion mode ESI-MS/MS analyses would be required to distinguish between sulfation or phosphorylation at Y 45 [83][84][85] Analysis of the MS1 spectra of F 41 WKQY 45 VDGDQCE 54 peptides showed that Y 45 is largely unmodified in rFIX from both H1 and H2 bioreactors ( Supplementary Fig. S6B).…”