Elucidation of high micro-heterogeneity of an acidic-neutral trichotoxin mixture from Trichoderma harzianum by electrospray ionization quadrupole time-of-flight mass spectrometry.

The high micro-heterogeneity of an acidic-neutral trichotoxin mixture from T. harzianum, PC01, was elucidated using a modern tandem mass spectrometer equipped with an electrospray ionization source, a hybrid quadrupole-orthogonal accelerator and a reflectron time-of-flight analyzer. The trichotoxins appeared predominantly in six possible doubly charged pseudo molecular ions with three different adducts (H, Na and K) as [M + 2H](2+), [M + H + Na](2+), [M + H + K](2+), [M + 2Na](2+), [M + Na + K](2+) and [M + 2K](2+). The singly charged pseudomolecular ions, [M + H](+), [M + Na](+) and [M + K](+), occurred only in low abundance when the cone voltages were higher than 30 V. Additional singly charged fragments, b(12) and y"6 (complementary N- and C-terminal fragments), were obtained in high abundance using high cone voltages. The peak patterns of both singly and doubly charged molecular adducts revealed that this trichotoxin mixture contained several components having 6-7 molecular masses with a consecutive 14 u difference among members in the same molecular adduct series. Furthermore, well resolved isotopic peaks of every doubly or singly charged ions and their reproducible peak intensity allowed the identification of the mixing of acidic trichotoxins 1 u molecular mass heavier than the neutral counterparts in the sample. Tandem mass spectrometric (MS/MS) analyses of various singly charged b(12) and y"6 ions supported the sequence deduction of the major and minor components and also the position of Glu in the sequences of these acidic molecules. The setting of either low or high resolution of the quadrupole mass filter unit together with a suitable variation of the collision voltage for any MS/MS precursor were the tools for extracting a number of mixed components and obtaining the major and minor sequences of these precursor peaks. The nature of the MS/MS fragmentation and the data assignment of three major doubly charged ions, [M + 2H](2+), [M + K + H](2+) and [M + 2K](2+), are demonstrated. Eleven new sequences of both acidic and neutral trichotoxins are reported.