Ultrasensitive Characterization of Site-Specific Glycosylation of Affinity-Purified Haptoglobin from Lung Cancer Patient Plasma Using 10 mu m i.d. Porous Layer Open Tubular Liquid Chromatography-Linear Ion Trap Collision-Induced Dissociation/Electron Transfer Dissociation Mass Spectrometry

作者：Wang Dongdong; Hincapie Marina; Rejtar Tomas; Karger Barry L^*

来源：Analytical Chemistry, 2011, 83(6): 2029-2037.

DOI：10.1021/ac102825g

Site-specific analysis of protein glycosylation is important for biochemical and clinical research efforts. Glycopeptide analysis using liquid chromatography-collision-induced dissociation/electron transfer dissociation mass spectrometry (LC-CID) allows simultaneous characterization of the glycan structure and attached peptide site. However, due to the low ionization efficiency of glycopeptides during electrospray ionization, 200-500 fmol of sample per injections is needed for a single LC-MS run, which makes it challenging for the analysis of limited amounts of glycoprotein purified from biological matrixes. To improve the sensitivity of LC-MS analysis for glycopeptides, an ultranarrow porous layer open tubular (PLOT) LC column (2.5 m x 10 mu m i.d.) was coupled to a linear ion trap (LTQ) collision-induced dissociation/electron transfer dissociation mass spectrometer to provide sensitive analysis of N-linked protein glycosylation heterogeneity. The potential of the developed method is demonstrated by the characterization of site-specfic glycosylation using haptoglobin (Hpt) as a model protein. To limit the amount of haptoglobin to low picomole amount of protein, we affinity purified it from 1 mu L pooled lung cancer patient plasma. A total of 26 glycoforms/glycan compositions on three Hpt tryptic glycopeptides were identified and quantified from 10LC-MS runs with a consumption of 100 fmol of Hpt digest (13 ng of protein, 10 fmol per injection). Included in this analysis was the determination of the glycan occupancy level. At this sample consumption level, the high sensitivity of the PLOT LC-LTQ-CID/ETD-MS system allowed glycopeptide identification and structure determination, along with relative quantitation of glycans presented on the same peptide backbone, even for low abundant glycopeptides at the similar to 100 amol level. The PLOT LC-MS system is shown to have sufficient sensitivity to allow characterization of site-specific protein glycosylation from trace levels of glycosylated proteins.

• 出版日期2011-3-15
• 单位东北大学