Top‐down and bottom‐up characterization of nitrated birch pollen allergen Bet v 1a with CZE hyphenated to an Orbitrap mass spectrometer

Abstract

Tyrosine (Tyr) residues of the major pollen allergen of birch Betula verrucosa, Bet v 1a, were nitrated by peroxynitrite. This modification enhances the allergenicity. Modified tyrosines were identified by analyzing intact allergen variants in combination with top‐down and bottom‐up approaches. Therefore, a laboratory‐built sheath‐liquid assisted ESI interface was applied for hyphenation of CE to an Orbitrap mass spectrometer to localize individual nitration sites. The major focus was on identification of primary nitration sites. The top‐down approach unambiguously identified Tyr 5 as the most prominent modification site. Fragments from the allergen core and the C‐terminal part carried up to three potential nitration sites, respectively. Thus, a bottom‐up approach with tryptic digest was used as a complementary strategy which allowed for the unambiguous localization of nitration sites within the respective peptides. Nitration propensity for individual Tyr residues was addressed by comparison of MS signals of nitrated peptides relative to all cognates of homolog primary sequence. Combined data identified surface exposed Tyr 5 and Tyr 66 as major nitration sites followed by less accessible Tyr 158 whereas Tyr 81, 83 and 150 possess a lower nitration tendency and are apparently modified in variants with higher nitration levels.

Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation, methodology and applications

“…Gusenkov et al. demonstrated the potential of combining a top‐down and a bottom‐up strategy in a complementary manner . The birch pollen allergen Bet v 1a was nitrated with peroxynitrite which resulted in the formation of nitrotyrosine residues, known to be responsible for an enhanced allergenicity.…”

Oligomerization and tyrosine nitration enhance the allergenic potential of the birch and grass pollen allergens Bet v 1 and Phl p 5

“…Y5 and Y120 nitration could not be detected and Y66 nitration was only found for ONOO -modified Bet v 1, but could not be quantified. Gusenkov and Stutz ( 60 ) later found that the reaction with ONOO leads to nitration of six tyrosine residues (Y5, Y66, Y81, Y83, Y150, Y158) with preferential nitration of the two surface-exposed tyrosine residues Y5 and Y66.…”

Peptide mapping of proteins by capillary electromigration methods

“…Both bottom-up peptide mapping and top-down proteomic approach have been applied for detailed characterization of nitrated birch pollen allergen Bet v 1a by CZE coupled to an Orbitrap MS detector using a home-made low-sheath-flow CZE-ESI interface [267]. The bottom-up peptide mapping approach with tryptic digest made possible the unambiguous localization of nitration sites on the particular Tyr residues.…”