Byonic annotates key elements of the spectrum, including the most commonly observed ion series for the type of fragmentation (a, b, y, c, z, b++, y++, etc.), common neutral losses from the precursor (e.g., M–98 for loss of phosphoric acid), immonium ions for certain amino acids, and oxonium ions for glycans.
Byonic also annotates key diagnostic peaks such as imm_pY at 216 Da for phosphotyrosine, 243 for phopho-Hex, and 284 for phospho-HexNAc. For Byonic, M alone refers to a precursor in its original charge state, so M+e in an ETD spectrum represents a charge-reduced precursor, and M_2+ – HexNAcHex in a spectrum of a plus-three precursor represents the loss of HexNAc, Hexose, and a proton.
In a glycopeptide spectrum, Pep refers to the neutral (possibly modified) peptide without glycans. For example, Pep+HexNAc_2+ represents the peptide with HexNAc and two protons, that is, the Y1++ ion.
Most of Byonic’s annotations of product ions are standard, but a few of the conventions may require additional explanation. If Byonic does not indicate charge, the charge is plus one. If Byonic does not indicate isotopes, the peak is the monoisotopic peak. For high-mass fragments, Byonic will append annotations to identify higher-isotope peaks; for example, _iso1, _iso2.
Common neutral losses are shown by removed nominal masses, for example, –17 = ammonia, –18 = water, –36 = two water molecules, –54 = three water molecules (a common loss from glycated lysine), –80 = loss of SO3 from sulfation, and –98 = loss of H3PO4 from phosphorylation. An apostrophe before a z-ion, for example, ‘z6 shows loss of (alkylated) side-chain on an N-terminal cysteine. A tilde shows loss of labile modifications, for example, ~y7 means y7 with loss of all labile modifications.
Labile modifications are defined to be O- and N-linked glycosylation, sulfation, histidine phosphorylation, and gamma-carboxylation. (In order to accommodate all the various types and modifications of O-linked glycans, Byonic treats any sufficiently heavy modification on S/T as labile.) For GalNAc- or GlcNAc-initiated glycosylation, Byonic will annotate peaks with a single base HexNAc, for example, ~y7+203.
Byonic annotates oxonium ions from glycans, either with names like “NeuAc–18” or atomic formulas when names are not definitive. The Byonic Viewer also annotates peaks at 284 and 446 for sulfated HexNAc and sulfated HexNAc-Hex. These are small peaks and they do not always show clearly.