Glycopeptides from human lactoferrin, which carries fucosylated and sialylated structures, served as positive controls; glycopeptides from invertase, which carries high-mannose structures, served as negative controls. Lactoferrin (A-C) and invertase (D-F) glycopeptides were affinity selected on AAL-conjugated POROS. Flow-though (FT) and bound (BD) fractions were collected and analyzed by MALDI-MS or MALDI-MS/MS. (A) MS spectrum of the lactoferrin FT1 fraction. (B) MS spectrum of the BD1 fraction. Peaks corresponding to various fucosylated glycoforms of three related peptides were observed (1) TAGWNVPIGTLRPFLNWTGPPEPIEAAVAR, (2) PFLNWTGPPEPIEAAVAR, and (3) LRPFLNWTGPPEPIEAAVAR; where N corresponded to glycosylated Asn with NWT as the consensus glycosylation sequence. (C) The BD1 fraction was rechromatographed and subjected to MS analysis; consistent with the initial results, glycopeptides were detected in the BD2 but not in the FT2 fraction (data not shown). The peptide backbone for these structures was confirmed by MS/MS. Monosaccharide composition and, in some cases, partial sequence information was also obtained for the carbohydrate structures (Supplementary Table 1 and Supplementary Figs. 1A-1G). These data (and the depicted glycans) were consistent with previously identified lactoferrin carbohydrate structures, and the fact that this molecule is highly fucosylated [40; 41]. (D) MS spectrum of the invertase FT1 fraction. Peaks corresponded to high-mannose structures appended to peptide “4” (AEPILNISNAGPWSR). (E) MS spectrum of the BD1 fraction. The glycopeptides were also observed but at approximately 10-fold less intensity. (F) The FT1 fraction was rechromatographed and subjected to MS analysis. The four molecular ions, which were also observed in the FT1 fraction (compare panels D and F), were interrogated by MS/MS and the peptide backbone and monosaccharide composition were confirmed (Supplementary Table 1 and Supplementary Figs. 1H-1K). These data (and the depicted glycans) are consistent with the fact that invertase presents high-mannose structures [42]. No invertase glycopeptides were detected in the rechromatographed BD2 fraction (data not shown). (■, N-acetylglucosamine; ●, galactose; ○, mannose; △, fucose; ◆, sialic acid.)

Positive (lactoferrin) and negative (invertase) glycoprotein standards were trypsin digested (1) and spiked into MARS 14-depleted, trypsin-digested human plasma (2). Then, the sample was subjected to lectin chromatography (3) on immobilized SNA (which binds sialic acid) or AAL (which binds fucose). The flow-through fraction (3a) contained peptides and glycopeptides not recognized by the lectin column. After a wash step (3b), elution buffer was applied and the bound fraction (3c) containing sialylated or fucosylated peptides and non-specifically bound species was collected. N-glycosylated peptides were deglycosylated with PNGase F (4), a peptidase that removes the intact glycan via cleavage of the amide bond on the asparagine side chain. This results in a +1 Da mass shift (indicated by an *) that is detectable by MS. Importantly, N-glycans are restricted to Asn residues within the sequence NXS/T, where X is any residue except proline. Thus, the presence of both features-the consensus sequence and mass shift-indicates that a peptide was formerly glycosylated.

(A) Survival curve depicting the number of AAL lectin chromatography experiments (x-axis) in which a given percentage of all glycosites (y-axis) were observed. (B) Box-whisker plot of the percentage overlap between all the possible pairs of datasets. The AAL and SNA datasets had 14×13/2 = 91 and 10×9/2=45 pairs to compare, respectively. For a pair of data sets the overlap was computed as |A ∧ B| / |A ∪ B|. For example, if one of the pairs consists of 154 and 151 glycosites respectively, with 141 in common, then the percentage overlap would be 141/164 = 86%.

Venn diagrams depict the total number of glycoproteins (A) or unique glycosites (B) captured from MARS 14-depleted, trypsin-digested human plasma by AAL, SNA, or both lectins using the finalized method.

ESI-MS/MS spectrum resulting from CID on the molecular ion MH2 ²⁺, selected at m/z 806.91 ²⁺ (M=1611.80). The spectrum was searched using Protein Pilot and peptide EVYPWYN*LTVEAK was identified (N* = deamidated N) with a peptide confidence of 99. Fragment ions y6 and y7 clearly show an increment mass difference of ΔM = 115 Da, localizing the deamidation to N442, the glycosite. Similar MS/MS spectra for all other glycosylated peptides are provided in Supplementary Fig. 2.