唾液酸化在許多細胞的功能表現中扮演一個很重要的角色。由於低含量、唾液酸醣胜肽離子低游離效率以及在純化的過程中，常常會發現唾液酸會脫離醣胜肽，因此用來提升純化唾液酸醣蛋白專一性以及偵測極限的分析工具成為了學者們感興趣的題目。在這邊，我們使用了正電荷基團裸露在材料外側的兩性離子親水層析管柱(ZIC-cHILIC)來純化唾液酸醣胜肽，希望能夠提升正電荷基團與帶負電荷的唾液酸之間的電子交互作用力進而提升純化的效率。藉由使用兩性親水離子層析管柱搭配直接沖堤分析物的方法並且利用質譜儀進行偵測，我們分別使用65%、60%、55%、50%和40 % ACN 進行直接冲堤，鑑定到了117、104、108、 134、136和52條醣胜肽，其中含有82、76、86、97、95和37條唾液酸醣胜肽。另外，我們也進一步採取階段式冲堤分析物的方法，使用不同百分比的乙腈冲堤出不同梯度的分析物。我們透過了兩性親水離子層析管柱並且利用0%-70%的乙腈冲堤並結合五個梯度的分析物，可以將使用基質輔助雷色脫附游離飛行質譜儀偵測到的唾液酸醣胜個數共32條(質荷比區間在3000到7500)。除此之外，我們使用這個策略對非小型細胞肺癌細胞株之中的唾液酸醣蛋白體進行分析。在蛋白質體學的分析中，我們分別使用了MAGIC和Byonic軟體，分別鑑定到了751條、1349條唾液酸醣胜肽。不同類型的醣結構，分別由兩個、三個、四個分支組成，像是 (1) 分支中沒有岩藻醣或是末端有唾液酸醣修飾(2) 分支中有岩藻醣或是末端有唾液酸醣修飾(3) 分支中僅有末端唾液酸醣修飾。並且，在這些醣胜肽中包含了在非小型細胞肺癌細胞株中十分重要的蛋白質EGFR蛋白13個醣基化點中的9個。總括來說，我們使用了兩性離子親水層析管柱並且搭配階段式梯度冲堤法有效的提升了鑑定唾液酸醣胜肽的專一性，並且增加了蛋白質體的覆蓋率。

Sialylation plays important roles in many cellular functions. Due to the low abundance, low ionization efficiency of sialo-glycopeptides and frequently observed dissociation of sialic acid residues during enrichment processes, tools to enhance the enrichment specificity and detection sensitivity in LC-MS/MS are of great interest. Here, we apply ZIC-cHILIC that carries phosphorylcholine functional group where the positively charged amine group is exposed outside to be more accessible to enhance the electrostatic attraction of negatively charged carboxylic group of sialic acid to purify the intact glycopeptides. By using single step ZIC-cHILIC and Orbitrap Velos analysis, in total, 82, 76, 86, 97, 95 and 37 unique sialo-glycopeptide were identified among 117, 104, 108, 134, 136 and 52 unique glycopeptide in the 65%, 60%, 55%, 50%, 40 % ACN and 0% ACN /0.5% FA direct elution, respectively by using cHILIC. We further applied a stepwise elution strategy for fractionating sialoglycopeptides of different properties by different percentage of acetonitrile (ACN). Combining 5 fractions from 0-70% ACN elution through cHILIC fractionation, the number increase from 15 to a total of 32 intact sialo-glycopeptides (m/z from 3000 to 7500) from fetuin were observed in MALDI-TOF analysis. Furthermore, we applied this strategy to study the sialo-glycoproteome in non-small cell lung cancer cells (PC9 cells). On the proteome scale, we identified 751 and 1349 intact sialo-glycopeptides in PC9 NSCLC cells by MAGIC and Byonic software, respectively. Different glycan compositions located on bi-, tri- and tetra-antennary such as (1) antennary without any fucosylation or terminal sialylation; (2) antennary with core- or terminal fucosylation but without sialylation; and (3) antennary with terminal sialylation were identified in our strategy. Among these identified glycoproteins, 9 of 13 N-linked glycosylation sites of epidermal growth factor receptor (EGFR), one of important yet well-known marker in NSCLC cell, were identified by our strategy. In summary, using cHILIC stepwise fractionation demonstrated specificity to enrich intact sialo-glycopeptides and increase the coverage on proteome scale.

1. Nakamori S, Kameyama M, Imaoka S, Furukawa H, Ishikawa O, Sasaki Y, et al. Increased expression of sialyl Lewisx antigen correlates with poor survival in patients with colorectal carcinoma: clinicopathological and immunohistochemical study. Cancer Res 1993;53:3632–7
2. Hedlund M, Ng E, Varki A, Varki NM. Alpha 2-6-Linked sialic acids on N-glycans
modulate carcinoma differentiation in vivo . Cancer Res 2008;68:388–94.
3. Tanaka F, Otake Y, Nakagawa T, Kawano Y, Miyahara R, Li M, et al. Expression of polysialic acid and STX, a human polysialyltransferase, is correlated with tumor progression in non-small cell lung cancer. Cancer Res 2000;60:3072–80.
4. Schultz MJ, Swindall AF, Bellis SL. Regulation of the metastatic cell phenotype by sialylated glycans. Cancer Metastasis Rev 2012;31:501–18.
5. Picco G, Julien S, Brockhausen I, Beatson R, Antonopoulos A, Haslam S, et al. Over-expression of ST3Gal-I promotes mammary tumorigenesis. Glycobiology 2010;20:1241–50.
6. Dube, D. H. & Bertozzi, C. R. Glycans in cancer and inflammation — potential for therapeutics and diagnostics. Nat Rev Drug Discov 4, 477–488 (2005).
7. Seales EC, Jurado GA, Singhal A, Bellis SL. Ras oncogene directs expression of a differentially sialylated, functionally altered beta1 integrin. Oncogene 2003;22:7137–45
8. Sakuma K, Aoki M, Kannagi R. Transcription factors c-Myc and CDX2 mediate E-selectin ligand expression in colon cancer cells undergoing EGF/bFGF-induced epithelial-mesenchymal transition. Proc Natl Acad Sci U S A 2012;109:7776–81.
9. Zhao, J., Simeone, D. M., Heidt, D., Anderson, M. A. & Lubman, D. M. Comparative Serum Glycoproteomics Using Lectin Selected Sialic Acid Glycoproteins with Mass Spectrometric Analysis:  Application to Pancreatic Cancer Serum. J. Proteome Res. 5, 1792–1802 (2006).
10. Cumming, D. A. et al. Structures of asparagine-linked oligosaccharides of the glycoprotein fetuin having sialic acid linked to N-acetylglucosamine. Biochemistry 28, 6500–6512 (1989).
11. Larsen, M. R., Jensen, S. S., Jakobsen, L. A. & Heegaard, N. H. H. Exploring the Sialiome Using Titanium Dioxide Chromatography and Mass Spectrometry. Mol Cell Proteomics 6, 1778–1787 (2007).
12. Palmisano, G. et al. A Novel Method for the Simultaneous Enrichment, Identification, and Quantification of Phosphopeptides and Sialylated Glycopeptides Applied to a Temporal Profile of Mouse Brain Development. Mol Cell Proteomics 11, 1191–1202 (2012).
13. Yu, L., Li, X., Guo, Z., Zhang, X. & Liang, X. Hydrophilic Interaction Chromatography Based Enrichment of Glycopeptides by Using Click Maltose: A Matrix with High Selectivity and Glycosylation Heterogeneity Coverage. Chemistry - A European Journal 15, 12618–12626 (2009).
14. Takegawa, Y. et al. Simple separation of isomeric sialylated N-glycopeptides by a zwitterionic type of hydrophilic interaction chromatography. J. Sep. Sci. 29, 2533–2540 (2006).
15. Weber, G., von Wirén, N. & Hayen, H. Hydrophilic interaction chromatography of small metal species in plants using sulfobetaine- and phosphorylcholine-type zwitterionic stationary phases. J. Sep. Science 31, 1615–1622 (2008).
16. Green, E. D.; Adelt, G.; Baenziger, J. U.; Wilson, S.; Van Halbeek, H. J. Biol.
Chem. 1988, 263, 18253–18268.
17. Powell, A. K. & Harvey, D. J. Stabilization of Sialic Acids in N-linked Oligosaccharides and Gangliosides for Analysis by Positive Ion Matrix-assisted Laser Desorption/Ionization Mass Spectrometry. Rapid Commun. Mass Spectrom. 10, 1027–1032 (1996).
18. Jeffrey S. Rohrer, Gregg A. Cooper, & R. Reid Townsend Analytical Biochemistry 212, 7-16 (1993).
19. Shisheng Sun , Punit Shah , Shadi Toghi Eshghi , Weiming Yang , Namita Trikannad , Shuang Yang , Lijun Chen , Paul Aiyetan , Naseruddin Höti , Zhen Zhang , Daniel W Chan & Hui Zhang Nature Biotechnology 34, 84–88 (2016).
20. Bern, M., Cai, Y., and Goldberg, D. (2007) Lookup peaks: a hybrid of denovo sequencing and database search for protein identification by tandem massspectrometry. Anal. Chem. 79, 1393–1400.
21. Lynn, K.-S.; Chen, C.-C.; Lih, T.-S. M.; Cheng, C.-W.; Su, W.-C.; Chang, C.-H.; Cheng, C.-Y.; Hsu, W.-L.; Chen, Y.-J.; Sung, T.-Y.MAGIC: an automated N-linked glycoprotein identification tool usinga Y1-ion pattern matching algorithm and in silico MS2 approach. Anal.Chem. 2015, 87, 2466−2473.
22. Dube, D. H. & Bertozzi, C. R. Glycans in cancer and inflammation — potential for therapeutics and diagnostics. Nat Rev Drug Discov 4, 477–488 (2005).
23. Meuillet EJ, et al. Sialidase gene transfection enhances epidermal growth factor receptor activity in an epidermoid carcinoma cell line, A431. Cancer Res. 1999;59:234–240.
24. Campbell, T. M., Main, M. J. & Fitzgerald, E. M. Functional expression of the voltage-gated Na+-channel Nav1.7 is necessary for EGF-mediated invasion in human non-small cell lung cancer cells. J Cell Sci 126, 4939–4949 (2013).
25. Dall'Olio F., Malagolini N., di Stefano G., Minni F., Marrano D., Serafini-Cessi F. (1989) Increased CMP-NeuAc:Galβ1,4GlcNAc-Rα2,6 sialyltransferase activity in human colorectal cancer tissues. Int. J. Cancer 44, 434–439
26. Sata T., Roth J., Zuber C., Stamm B., Heitz P. U. (1991) Expression of α2,6-linked sialic acid residues in neoplastic but not in normal human colonic mucosa: A lectin-gold cytochemical study with Sambucus nigra and Maackia amurensis lectins. Am. J. Pathol. 139, 1435–1448.
27. Dall'Olio F., Malagolini N., Serafini-Cessi F. (1992) The expression of soluble and cell-bound α2,6-sialyltransferase in human colonic carcinoma CaCo-2 cells correlates with the degree of enterocytic differentiation. Biochem. Biophys. Res. Commun. 184, 1405–1410.
28. Gessner P., Riedl S., Quentmaier A., Kemmner W. (1993) Enhanced activity of CMP-neuAc:Galβ1–4GlcNAc:α2,6-sialyltransferase in metastasizing human colorectal tumor tissue and serum of tumor patients. Cancer Lett. 75, 143–149.
29. Murayama T., Zuber C., Seelentag W. K., Li W. P., Kemmner W., Heitz P. U., Roth J. (1997) Colon carcinoma glycoproteins carrying α2,6-linked sialic acid reactive with Sambucus nigra agglutinin are not constitutively expressed in normal human colon mucosa and are distinct from sialyl-Tn antigen. Int. J. Cancer 70, 575–581.
30. Skacel P. O., Edwards A. J., Harrison C. T., Watkins W. M. (1991) Enzymic control of the expression of the X determinant (CD15) in human myeloid cells during maturation: The regulatory role of 6-sialytransferase. Blood 78, 1452–1460.
31. Recchi M. A., Harduin-Lepers A., Boilly-Marer Y., Verbert A., Delannoy P. (1998) Multiplex RT-PCR method for the analysis of the expression of human sialyltransferases: Application to breast cancer cells. Glycoconj. J. 15, 19–27.
32. Wang P. H., Li Y. F., Juang C. M., Lee Y. R., Chao H. T., Tsai Y. C., Yuan C. C. (2001) Altered mRNA expression of sialyltransferase in squamous cell carcinomas of the cervix. Gynecol. Oncol. 83, 121–127.
33. Fukushima K., Hara-Kuge S., Seko A., Ikehara Y., Yamashita K. (1998) Elevation of α2->6 sialyltransferase and α1–>2 fucosyltransferase activities in human choriocarcinoma. Cancer Res. 58, 4301–4306.
34. Kaneko Y., Yamamoto H., Kersey D. S., Colley K. J., Leestma J. E., Moskal J. R. (1996) The expression of Galβ1,4GlcNAcα2,6 sialyltransferase and α2,6-linked sialoglycoconjugates in human brain tumors. Acta Neuropathol. 91, 284–292.
35. Nie, H., Li, Y. & Sun, X.-L. Recent Advances in Sialic Acid-Focused Glycomics. J Proteomics 75, 3098–3112 (2012).
36. Johnson LN, Barford D. The effects of phosphorylation on thestructure and function of proteins. Annu Rev Biophys BiomolStruct 1993;22:199–232
37. Hunter T. Signaling — 2000 and beyond. Cell 2000;100:113–27.
38. Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S.The protein kinase complement of the human genome.Science NY 2002;298 1912.
39. Roddick-Lanzilotta, A. D., and McQuillan, A. J. (2000) An in situ infraredspectroscopic study of glutamic acid and of aspartic acid adsorbed on
TiO2: implications for the biocompatibility of titanium. J. Colloid Interface
Sci.227,48–54.
40. A simple integrated system for rapid analysis of sialic-acid-containing N-glycopeptides from human serum - Zhu - 2013 – PROTEOMICS.
41. Linden JC, Lawhead CL (1975) J Chromatogr A 105:125–133.
42. Hagglund P, Bunkenborg J, Elortza F, Jensen ON, Roepstorff P (2004) J Proteome Res 3:556–566.
43. Alpert AJ, Andrews PC (1988) J Chromatogr 443:85–96.
44. Yu YQ, Fournier J, Gilar M, Gebler JC (2007) Anal Chem 79:1731–1738.
45. Takegawa, Y.; Deguchi, K.; Ito, H.; Keira, T.; Nakagawa, H.;Nishimura, S.J. Sep. Sci.
2006,29, 2533–2540.
46. Hägglund P1, Bunkenborg J, Elortza F, Jensen ON, Roepstorff P (2004) A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation. J Proteome Res. 2004 May-Jun;3(3):556-66.
47. Scott, N. E. et al. Simultaneous Glycan-Peptide Characterization Using Hydrophilic Interaction Chromatography and Parallel Fragmentation by CID, Higher Energy Collisional Dissociation, and Electron Transfer Dissociation MS Applied to the N-Linked Glycoproteome of Campylobacter jejuni. Mol Cell Proteomics 10, (2011).
48. Parker BL,et al. (2011) Quantitative N-linked Glycoproteomics of Myocardial Ischemia and Reperfusion Injury Reveals Early Remodeling in the Extracellular Environment. Molecular & Cellular Proteomics, 10.
49. Ono M, Hirata A, Kometani T, et al. Sensitivity to gefitinib (Iressa, ZD1839) in non-small cell lungcancer cell lines correlates with dependence on the epidermal growth factor (EGF) receptor/extracellular signal-regulated kinase 1/2 and EGF receptor/Akt pathway for prolifera. Mol CancerTher 2004;3(4):465–472
50. Thaysen-Andersen, M., Mysling, S. & Højrup, P. Site-Specific Glycoprofiling of N-Linked Glycopeptides Using MALDI-TOF MS: Strong Correlation between Signal Strength and Glycoform Quantities. Anal. Chem. 81, 3933–3943 (2009).
51. Eylar, E. H., Madoff, M. A., Brody, O. V. & Oncley, J. L. The Contribution of Sialic Acid to the Surface Charge of the Erythrocyte. J. Biol. Chem. 237, 1992–2000 (1962).
52. Serena Di Palma, Shabaz Mohammed. Zwitterionic Hydrophilic Interaction Liquid Chromatography (ZICHILIC and ZIC-cHILIC) Provide High Resolution Separation and Increase Sensitivity in Proteome AnalysisAnal. Chem. 2011, 83, 3440–3447