RaPID GLYcoPePtIDe EnrIcHment UsIng CeLLuLose HYDroPHILIc InteractIon/ReverseD-PHase StageTIPs

1 Laboratory of Biopharmaceutical and Regenerative Sciences, Graduate School of Medical Life Science, Yokohama City University, 1–7–29 Suehiro-cho, Tsurumi-ku, Yokohama 230–0045, Japan 2 Laboratory of Biopharmaceutical and Regenerative Sciences, Medical Life Science, Division of Sciences, International College of Arts and Sciences, Yokohama City University, 1–7–29 Suehiro-cho, Tsurumi-ku, Yokohama 230–0045, Japan


INTRODUCTION
Glycosylation of proteins is a common post-translational modi cation and plays an important role in the formation of a protein structure, quality control, signal transduction, and immune response. [1][2][3][4] Biologics, including monoclonal antibody (mAb) drugs, represents a successful and expanding class of drugs because of their high speci city and therapeutic e ects. 5) e production of mAb therapeutics requires careful monitoring of glycans because their antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) are dependent on their glycan structures. 6,7) For example, the α1-6 fucosylation of a non-reducing-end GlcNAc decreases ADCC activity, 6,8) whereas galactosyl modi cation increases CDC activity. 7) Since glycans a ect the safety and e cacy of biopharmaceuticals, they are considered to be critical-quality attributes in drug quality control. erefore, to elucidate biological phenomena and develop biopharmaceuticals, it is frequently necessary to quantitatively analyze protein glycosylation.
In general, analyses glycoprotein glycosylation can be classi ed into three mass-spectrometric approaches: using the intact glycoprotein, the released glycan, or a glycopeptide approach. 9,10) Intact glycoproteins can be directly subjected to mass spectrometry in the intact glycoprotein approach. Although in-depth sample preparation is not required in this approach, the method is not very sensitive. e released-glycan approach provides collecting detailed structural information regarding a glycan but does not reveal any glycosylation site or genetic information. e glycopeptide approach is a promising method for the glycosylation analysis of glycoproteins, through which it is possible to obtain comprehensive glycosylation information from complex biological samples. However, in mass spectrometry (MS), signals produced from glycopeptides are low compared to signals corresponding to peptides, owing to the low ionization e cacy of glycopeptides and the heterogeneity of glycans. us, glycopeptide enrichment is indispensable for analyzing glycosylation in glycoproteins using MS.
Several methods for enriching glycopeptides for MS have been reported, including lectin a nity chromatography and a batch method using a hydrophilic interaction liquid chromatography (HILIC) resin. Although lectin chromatography can be used to classify and enrich glycopeptides based on the speci cities of lectins to glycan epitopes, it cannot cover all types of glycans, and the method is a costly one. e batch method typically involves the use of cellulose and agarose resins and is a promising method for glycopeptide enrichment. 11,12) However, the method is time-consuming, and the method cannot be applied to small amounts of sample. e stop-and-go extraction tip (StageTip) has been widely used in sample pre-puri cation for MS. StageTips have become extremely popular in proteomics because of their low price, high throughput, high recovery rate for small amount samples, and ease of use. 13) We report herein on a novel StageTip prepared by combining cellulose HILIC and reversed phase (RP) and its use in e ciently enriching glycopeptides in a sample. e developed StageTip method allows for the rapid and convenient enrichment of a small sample that contains both N-and O-glycosylated peptides.

Digestion
IgG1 aliquots (50 µg) the α1-acid glycoprotein, hCG, and RNase B were dissolved in 50 µL 0.1% (w/v) RapiGest SF containing 50 mM Tris-HCl (pH 7.8). An aliquot (0.5 µL) of 500 mM DTT was added to each sample, and the samples were then incubated at 65°C for 30 min. e samples were alkylated by adding 1.4 µL of 500 mM IAA, followed by incubation at room temperature for 40 min in the dark. Alkylation was terminated by adding 0.2 µL 500 mM DTT. Trypsin Gold aliquots (2 µg) were added to the IgG1, hCG, and α1-acid glycoprotein samples. A LysC aliquot (2 µg) was added to the RNase B sample. All samples were incubated at 37°C for 16 h. RapiGest SF was denatured and removed from the samples as indicated in the manufacturer's manual. e samples were desalted using Sep-Pak Vac 1cc C18 cartridges and dried using a speed-vac.
e N-glycans in the hCG were released by treatment with 1 U N-glycosidase F (Roche Diagnostics, Rotkreuz, Switzerland) in 50 µL 250 mM sodium phosphate bu er (pH 7.5) for 16 h at 37°C. e hCG sample was desalted using Sep-Pak Vac 1cc C18 cartridges and dried via a speed-vac. Finally, the samples were reconstituted with 50 µL 0.1% formic acid.

Preparation of cellulose-RP StageTip
Microcrystalline cellulose was washed with water eight times and then washed twice with an 80% acetonitrile/0.1% TFA (v/v) solution. e microcrystalline cellulose was suspended in 80% acetonitrile (v/v). A repeating pipette tip (Distritip mini 1250 µL; Gilson, Middleton) was cut about 5 mm from the tip end. e suspension of microcrystalline cellulose was drawn in the repeating pipette tip and allowed to stand for 30 s to permit the microcrystalline cellulose to precipitate. Aliquots (25 µL) of the microcrystalline-cellulose slurry in 80% acetonitrile were placed in the StageTips (C-TIP, Nikkyo Technos). Finally, the StageTips were centrifuged in a dedicated tabletop centrifuge for 20 s. We repeated the experiment using di erent lling conditions. e optimal conditions for lling microcrystalline cellulose owing to its surface tension and viscosity was 80% acetonitrile.

Glycopeptide enrichment by the cellulose-RP StageTips
e cellulose-RP StageTips were inserted in the rotor of a dedicated tabletop centrifuge. Aliquots (50 µL) of 0.1% TFA were placed in the StageTips and centrifuged for 30 s to wash the cellulose resin. Aliquots (50 µL) of 70-90% acetonitrile/0.1% TFA (sample loading solution: 80% for N-linked glycopeptides and 90% O-linked glycopeptides; optimized condition) were then placed in the StageTips and centrifuged for 20 s for initialization of the microcrystalline cellulose. Aliquots (50 µL) of the sample loading solution were placed in the StageTips and ashed for 1 s. Samples (1 µg protein digested in 1 µL 0.1% formic acid/water) were placed in the solution and centrifuged for 20 s to allow them to bind to the cellulose layer (Fig. 1G). Aliquots (100 µL) of 70-90% acetonitrile/0.1% TFA (selective enrichment solution: 80% for N-linked glycopeptides and 90% O-linked glycopeptides; optimized condition) were placed in the StageTips and centrifuged for 20 s; this procedure was repeated three times to ensure the nonspeci c peptides were completely removed (Fig. 1H). Aliquots (50 µL) of Milli-Q water were placed in the StageTips, and these were centrifuged for 30 s for re-enrichment of glycopeptides RP layer. Finally, aliquots (20 µL) of 80% acetonitrile were placed in the StageTips, and the glycopeptides were eluated using a dedicated syringe (Figs. 1E and 1F).

Intensity ratio calculation
Glycopeptide-intensity ratios a er enrichment were calculated by comparing the peak areas in the extracted-ion chromatograms of glycopeptides before and a er enrichment. e peak areas in the extracted-ion chromatograms were calculated using the Xcalibur so ware (Xcalibur 2.2; ermo Fisher Scienti c).

Enrichment of N-glycosylated peptides
e enrichment procedure involves four steps: loading a glycoprotein digest on the StageTips, selective enrichment of the glycopeptides on HILIC, re-enrichment of the glycopeptides on RP, and elution of the glycopeptides from the StageTips (Fig. 1). For the e ective absorption of the Nglycosylated peptides on HILIC, various acetonitrile/TFA solutions (70-90% acetonitrile/0.1% TFA) were prepared for use with 10-25 µL of the microcrystalline cellulose.
e results indicate that using 80% acetonitrile, but not the volume of the microcrystalline cellulose, is crucial for the HILIC enrichment process. Figures 2A-1 and 2B-1 show total ion current (TIC) chromatograms of a LysC digest prepared from RNase B, which contains a high-mannose type glycan, before and a er enrichment, respectively. e undetectable peaks at 7-8 min in Fig. 2A-1 are detectable a er enrichment with the StageTips composed of cellulose (25 µL) and RP (Fig. 2B-1). e full mass scans of non-enriched glycopeptides (Fig. 2B-1) and enriched glycopeptides (Fig. 2B-2) as well as the intensity ratios of glycoforms (Table 1) indicate that the peak intensities of the glycoforms are increased a er enrichment, and the peak enhancement e ect is more notable in the case of lesser glycoforms.
is phenomenon can be explained by the reduction in non-glycosylated peptides that causes the ionization of glycopeptides to be suppressed, resulting in less intense peaks. Figure 3 shows the e ect of enriching the α1-acid glycoprotein, which contains biantennary, triantennary, and tetra-antennary glycans. A er enrichment, many peptide peaks disappear, and glycopeptide peaks (GP-A, B, C, and D) become detectable (Figs. 3A-1 and 3B-1). However, the intensity of glycoform ion peaks of GP-A at 9.3-9.6 min is reduced a er the enrichment (Figs. 3A-2 and 3B-2, Table 2). In contrast, several glycoform ion peaks of GP-B at 15.0-16.4 min become more intense a er enrichment (Figs. 3A-3 and 3B-3). e amino acid sequence of GP-A is NEEYNK, which is relatively hydrophilic. e glycopeptides GP-A pass through the RPlayer during re-enrichment with H 2 O because 80% of the acetonitrile solution remains in the cellulose layer. e cellulose/RP StageTips are suitable for enriching glycosylated peptides consisting of hydrophobic amino acid residues.
e TIC chromatograms of non-enriched and enriched IgG1 glycopeptides (Figs. 5A-1 and 5B-1), as well as full mass scans of GP-A at 10.0-11.0 min (Figs. 5A-2 and 5B-2), revealed that the Stage Tip is relatively selective. However, the intensity ratios of glycoforms are almost 100%, suggesting that the glycopeptides are not completely adsorbed on the RP layer. Based on the results for RNase B and α1-acid glycoprotein, su cient enrichment of glycopeptides tends to enhance the peak intensities of glycopeptides by reducing the level of ionization suppression. us, we measured the ow through fractions a er loading an IgG1 tryptic digest (1 µg of 1 µg/µL). Compared with the peak intensity of non-enriched G0F (Fig. 6A-2), the intensity of G0F is higher a er sample loading ( Fig. 6D-2) and selective enrichment (Fig. 6E-2). We estimate that about 60% of the glycopeptides are leaked in the re-enrichment step, and 30% is recovered in the elution step. For the enrichment of hydrophilic glycopeptides (e.g., IgG1 glycopeptides), the ow-through fraction, which is obtained a er re-enrichment, must be combined with the eluted fraction and then dried for maximizing the recovery ratios. It is noteworthy that the peak intensities of the respective Table 1. Intensity ratios of glycopeptides of RNase B a er/before enrichment.
"Pep" indicates the amino acid sequence SRNLTK. (mean±S.D., n=4) Table 2. Intensity ratios of glycopeptides of α1-acid glycoprotein a er/before enrichment. glycoforms are nearly the same, suggesting that the distribution of glycoforms does not change a er the StageTip treatment (Table 3). Fucose and galactose residues in glycans on mAbs a ect both ADCC and CDC. erefore, glycans on mAbs showing ADCC and CDC should be controlled and monitored in the manufacturing process. Our cellulose-RP StageTips method is suitable for monitoring mAbs glycans, because it is easy, disposable, glycoform independent, and has a carry-over free format.

Enrichment of O-glycosylated glycopeptides
We next attempted to enrich the O-linked glycopeptides (peak area ratio, Table 4). Because O-linked glycans are smaller than N-linked glycans, a higher acetonitrile concentration (90% acetonitrile/0.1% TFA) is used for enriching O-glycosylated glycopeptides than for N-glycosylated glycopeptides. Before enrichment, the observed glycopeptide peaks are minor (Fig. 7A-1), whereas, a er enrichment, they are major (Fig. 6A-2). Although the enrichment of O-  glycosylated glycopeptides is not as e ective as the enrichment of N-glycosylated glycopeptides, owing to the nonspeci c absorption of peptides, we conclude that this method can also be used to enrich O-glycosylated glycopeptides.

CONCLUSION
We report on the development of a method for enriching small amounts of glycopeptides using cellulose reversedphase StageTips. Enrichment of N-linked and O-linked glycopeptides can be achieved at the micro to nanogram level within a few minutes.   Table 3. Intensity ratios of glycopeptides of IgG1 a er/before enrichment.