APPLICATION NOTE
Byonic™: N-Linked Glycopeptide Analysis
Marshall Bern, Ph.D., Protein Metrics Inc. (www.proteinmetrics.com)
October 2014
Benefits summary


Best practices for optimizing an N-linked glycopeptide search.
How-to for using Byonic to effectively search for N-linked glycopeptides
Method
N-linked glycosylation may be the most common, and is certainly the most complex, of all
posttranslational modifications of proteins. It is also one of the most difficult to study, due
to its extreme complexity and the weaker ionization and fragmentation of glycopeptides
relative to ordinary peptides.
Byonic identifies glycopeptides to the level of peptide
sequence and glycan composition. A glycan
composition is given by a string such as
HexNAc(4)Hex(5)Fuc(1)NeuAc(2), which specifies the
monosaccharide composition, but does not distinguish
isomers such as GlcNAc and GalNAc, nor identify the
branching structure (called “topology” or “cartoon”)
Figure 1. A possible structure for
and linkage information (positions and stereochemistry HexNAc(4)Hex(5)Fuc(1)NeuAc(2).
of the glycosidic bonds). In this particular case,
however, there is one most likely topology, and less
than 10 combinations of linkage types for that topology. See Figure 1. We limited Byonic
to the composition level, because topology and especially linkage are rarely discernible
from a glycopeptide mass spectrum alone.
Byonic’s input user interface includes a tab called Glycans. Clicking on Enter/Edit on the
Glycans tab brings up the box shown in Figure 2, which offers three different ways to set
glycan modifications:
Application Note - Byonic™: N-linked glycopeptide analysis
1
APPLICATION NOTE
Ways to set glycan modifications
1. The top section entitled Enter glycan database(s) allows the user to input a glycan
database in a simple text format. (Several useful glycan databases are provided with
the program. These can be modified or you can create your own!) The user selects
N-Glycan or O-glycan for each database, the name of the glycan database, and the
maximum number of instances of each glycan per peptide and whether those
instances count against the Total common max or Total rare max limit on the
Figure 2. Byonic provides three ways to set glycan modifications: glycan databases (top, with
pull-down menu open), specific glycan setting from a menu (middle with pop-up box open), and
free text input (bottom). A glycan modification is defined by composition, potential site, and fine
control (rare1, common2, etc.); top, middle, and bottom are just different input “grammars”.
Application Note - Byonic™: N-linked glycopeptide analysis
2
APPLICATION NOTE
Modifications tab. Setting a glycan database to rare1 with Total rare max set to 1
means that peptides with two glycans will not be considered, but setting the
database to common1 with Total common max set to 2 means that peptides with
two different glycans will be considered and may account for most of the search
time. Our recommended best practice is to searching N-glycan databases using
rare1 unless you have a good reason to do otherwise.
Each line of a glycan database gives a glycan composition such as
HexNAc(2)Hex(2)Fuc(10)NeuGc(1)Sulfo(1). The order of the monosaccharides
does not matter, and the glycan need not be biologically possible. Byonic supports a
long list of keywords including HexNAc, Hex, Fuc, dHex, NeuAc, NeuGc, Pent, GlcA,
IdoA, DiNAcBac, Acetyl, Sulfo, Phospho, Na, and Sodium.
2. The middle section entitled Enter specific glycan(s) allows the user to enter glycans
one at a time. Clicking on the button labeled … brings up a menu of the six most
common monosaccharides (HexNAc, Hex, Fuc, Pent, NeuAc, NeuGc) along with
Sodium and a box labeled Additional mass for mass deltas currently unknown to
Byonic such as glycan derivitizations or adducts.
3. The bottom section entitled Enter custom glycan text in fine control format allows
the user to cut and paste glycan definitions and setting rare and common
individually instead of loading an entire file. The rules are in the same format
Byonic uses for ordinary modifications, for example,
HexNAc(2)Hex(2)Fuc(1) @ NGlycan | common1
sets a paucimannose N-glycan on the NX{S/T} motif, where X is any residue except
proline. NGlycan specifies the N-glycosylation motif, and OGlycan specifies S or T.
One can use these keywords with other modifications, for example,
Delta:H(1)O(-1)18O(1) / +2.988261 @ NGlycan | common1
specifies deglycosylation in 18O water only at asparagines in the motif. Conversely,
one can also write
HexNAc(2)Hex(2)Fuc(1) @ N | common1
to search for an N-glycan on any asparagine to pick up rare cases such as Nglycosylations on NXC or on reverse motifs.
Application Note - Byonic™: N-linked glycopeptide analysis
3
APPLICATION NOTE
Example
We obtained data from blood plasma enriched for glycopeptides with wheat germ
agglutinin, reduced with DTT, digested with trypsin, and alkylated with iodoacetamide. We
searched the data for fully tryptic peptides with at most one missed cleavage, and used 10
ppm precursor tolerance for Orbitrap MS1 and 0.5 Da fragment tolerance for ETD ion-trap
MS2. We used a small database containing about 200 abundant plasma proteins, including
all the most abundant glycoproteins. We set the glycan modifications as shown in Figure 2:
the database N-glycan 57 human plasma, included in the Byonic download, was set to
rare1, and 7 common1 glycans were entered individually to allow for two-glycan peptides
containing one of these 7 along with one of the 57 rare1 glycans. This gives a slightly faster
search than setting N-glycan 57 human plasma to be common1 or common2.
Byonic found about 35 glycoproteins in this small data set (2733 MS2 spectra), with
multiple glycosylation sites and multiple glycans (up to about ~10 distinct compositions)
per site. See Figure 3.
Figure 3. Glycopeptide search results as viewed in Byonic. Notice the strong loss of NeuAc
from the charge-reduced precursors in the displayed spectrum.
Protein Metrics Inc.
San Carlos, CA
www.proteinmetrics.com
Application Note - Byonic™: N-linked glycopeptide analysis
4