Mass Spectral Library Methods for Analysis of Site-Specific N‑Glycosylation: Application to Human Milk Proteins

We present a mass spectral library-based method for analyzing
site-specific
N-linked protein glycosylation. Its operation and utility are illustrated
by applying it to both newly measured and available proteomics data
of human milk glycoproteins. It generates two varieties of mass spectral
libraries. One contains glycopeptide abundance distribution spectra
(GADS). The other contains tandem mass spectra of the underlying glycopeptides.
Both originate from identified glycopeptides in proteolytic digests
of human milk and purified glycoproteins, which include tenascin,
lactoferrin, and several antibodies. Analysis was also applied to
digests of a NIST human milk standard reference material (SRM), leading
to a GADS library of N-glycopeptides, enabling the direct comparison
of glycopeptide distributions for individual proteins. Tandem spectra
underlying each glycopeptide GADS peak are combined to create a second
type of library that contains spectra of the underlying glycopeptide
spectra. These were acquired by higher-energy (stepped) collision
dissociation fragmentation followed by ion-trap fragmentation. Spectra
are annotated using MS_Piano, recently reported annotation software.
This data, with extensions of a widely used spectral library search
and display software, provides accessible mass spectral libraries.