T cell/transmembrane immunoglobulin and mucin protein-1 or TIM-1 is expressed on the surface of T cells and plays a role in tumor immunity. Containing a mucin domain spanning residues 109 to 259, a 151-residue section in total, TIM-1 has 67 potential glycosites, meaning just under 45% of the amino acids are serine or threonine residues. Most of these glycosites are adjacent or proximal to one another; in some cases, four sites exist sequentially. Thus, TIM-1 represents a challenging benchmark protein for characterization. After SmE digestion, 691 glycopeptides were separated by nanoLC and identified and characterized by HCD, EThcD, and UVPD. Examples of the MS/MS spectra are shown for one representative peptide in Figure (HCD and EThcD) and Figure (with additional ions labelled in Figure ). Consistent with prior reports, HCD primarily generates b-and y-type ions and offers limited structural information about the glycan. EThcD produces a more extensive array of fragment ions, including c-and z-type ions, and is known for localizing O-glycosites because the glycans may be preserved on the resulting fragment ions. UVPD yields peptide sequence ions, including ones that retain the entire glycan or partial glycan, in addition to glycan ions which reveal compositional information. For each glycopeptide, the peptide sequence and glycan composition at each site were identified using Byonic.