TABLE 3.
Lectin dispersive solid phase extraction using commercial sorbents.
| Lectin/provider | Affinity sorbent amount | Sample, sample volume or amount | Processing before and during the extraction on lectin | Washing conditions | Desorption conditions | Processing after lectin desorption | Objective | References |
|---|---|---|---|---|---|---|---|---|
| Glycoproteins | ||||||||
| AAL, SNA/Vector labs | 60 μl SNA and 90 μl AAL | human serum | Depletion, incubation: overnight at 4°C | 50 mM PBS pH 7.5, 200 µl (x5) | 100 mM acetic acid, 400 µl | tryptic digestion, LC-MS/MS | Comparison of lectin enrichment with hydrazide chemistry for biomarker discovery | Song et al. (2014) |
| AAL, WGA, SNA/Vector labs | intact proteins: 250 μl lectin sorbent glycopeptides: 100 μl lectin sorbent | human serum, 25 μl | dilution at 1:10 in buffer, incubation: overnight at 4°C | 200 mM Tris buffer pH 7.5 (x3) | AAL: 0.1 M Fuc SNA: 0.5 mM Lac WGA: 0.8 M N-GlcNAc in 0.2 M acetic acid, 100 μl, 30 min | tryptic digestion, second lectin enrichment, hydrazide enrichment, PNGase F LC–MS/MS | comparison of three approaches for enrichment of the glycoproteome of human sera | Li et al. (2015) |
| Con A, WGA, Jacalin/Vectorlabs | mix of 200 μl | human follicular fluid | incubation: overnight at 4°C | PBS (1x) | Con A: 0.2 M α-MM WGA: 0.5 M GlcNAc JAC: 0.1 M Mel, 30 min incubation, 10 min centrifugation at 4°C (x2) | tryptic digestion, labelling, SCX, SAX stage tip fractionation, LC-MS/MS, immunoblotting | glycoproteome study for biomarker discovery | Patil et al. (2019) |
| VVA | N.S | organelles from Toxoplasma gondii | parasite lysate in RIPA buffer | 50 mM Tris pH 7.4 + metal ions (x5) | GalNAc | SDS-PAGE, in-gel tryptic digestion, MS | Identification of O-glycoproteins and assessment of their function | Wang et al. (2016) |
| VVA/Vector labs | 40 µl | protein precipice of cell extract, 600 mg | incubation: overnight at 4°C | 10 mM HEPES pH 7.5 + metal ions (x3) | boiling (100 °C, 8 min) in Laemmli buffer+ 5% 2BME, 1 mM EDTA, 80 µl | 1) SDS-PAGE 2) lectin blotting 3) in-gel tryptic digestion, LC-MS and MS/MS | Characterization of Tn-modified glycoproteins | Hoja-Łukowicz et al. (2018) |
| Glycopeptides | ||||||||
| LCH/GE Healthcare | 100 μl | mouse liver tissue, 1 mg HeLa cells, 100 µl (tryptic digest) | tryptic digestion, HILIC, incubation: overnight at 4°C | 20 mM Tris- pH 7.45 + metal ions, 400 µl (x4) | 1 M acetate, 150 μl, 15min elution (x3) | Endo F3, LC-MS/MS | HILIC and lectin enrichment followed by fragmentation and spectrum refinement method, for the analysis of core fucosylation | Cao et al. (2014) |
| LCA, PSA, AAL LTL, UEA I/Vector labs AOL-biotin conjugate/TCI America | 150 µg/overnight at 4°C | cell culture, 150 µg (tryptic digest) | tryptic digestion incubation: overnight at 4°C | TBS pH 7.4 + metal ions/0.5 ml (x4) | LCA and PSA: 0.2 M α-MM and0.2 M α-MG AAL, LTL, UEA I and AOL: 0.1 M Fuc, 0.5 ml | hSAX LC-MS/MS | analysis of the fucome by combination of enrichment with lectins and hSAX | Zhou et al. (2017) |
| Con A/GE Healthcare | 150 µl | Leafs from Zea mays L. Plant, 0.5 mg | Tryptic digestion, labelling incubation: 1 h | 10% ACN, 50 mM ammonium bicarbonate, 300 µl (x5) | 0.5 M Man, 75 µl (x2) | PNGase A + PNGase F, LC-MS/MS | N-glycoproteome analysis | Bu et al. (2017) |
NOTES: hSAX: hydrophilic strong anion exchange chromatography; N.S: not specified; PBS: phosphate buffered saline; RIPA: radioimmunoprecipitation; SAX: strong anion exchange chromatography; SCX: strong cation exchange chromatography TBS: tris-buffered saline.