| dc.description.abstract | Among various protein post-translational modification (PTM), N-linked glycosylation and phosphorylation are among the most ubiquitous PTMs and play important roles in many biological functions, such as signal transduction, protein stability, and mobility. However, due to inherent heterogeneity of modification site and structure and ion suppression effect from free peptides during mass spectrometry analysis, it is necessary to develop enrichment strategy prior to mass spectrometry (MS) analysis. Though there are many enrichment methods developed for these two PTMs, both glycopeptides and phosphopeptides require different enrichment approaches.
Recently, magnetic nanoparticles (MNPs) have been widely used in biomedical applications mainly because of its capability for surface functionalization and fast separation by magnetic extraction. In this thesis, based on the hydrophilic interaction, active NH2- and zwitterionic functional groups were anchored (provided by Elmer Austria Jr.) on the surface of the magnetic nanoparticles (amine/zwitterionic-hydrophilic interaction based magnetic nanoparticle, NH2/ZIC-HILIC MNP) for simultaneous enrichment of glycopeptides and phosphopeptides. Four main parts were optimized in this work, including buffer composition, pH value, organic composition and particle amount. In glycopeptide enrichment, 0.1% formic acid (FA) with 95% acetonitrile (ACN) (adjusted to pH 7) were used as incubation buffer; 1% phosphoric acid with 80% ACN and 0.1% ammonium hydroxide (NH4OH) with 80% ACN were applied as washing buffer and 2% NH4OH/50% ACN for elution buffer respectively.
On the other hand, 0.1% FA/95% ACN (adjusted to pH 3) were used as incubation buffer. 6% acetic acid (AA) with 85% ACN and 5% NH4OH/50% ACN were applied as washing buffer and elution buffer respectively. 2μg peptides to 100μg nanoparticles was the optimized usage ratio for both glycopeptide and phosphopeptide enrichment.
By pH modulation strategy, effective separation of both glycopeptides in pH=7 and phosphopeptides in pH=3 can be achieved from a 1:1:1 mixture of α- and β-casein (phosphoproteins) and HRP (glycoprotein), 10 phosphopeptides in phosphopeptide elute fraction and 9 glycopeptides in glycopeptide elute fraction were identified in MALDI-TOF analysis with 96.3% and 81.1% separation efficiency for phosphopeptides and glycopeptides respectively.
To increase the complexity of the sample, bovine serum albumin (BSA) protein was added into the sample. In ratio of HRP : α-casein : β-casein : BSA= 1:1:1:1, 8 phosphopeptides still can be detected and dominate in the phosphopeptide elute fraction; in the glycopeptide elute fraction, though there are still 7 glycopeptides can be detected, When BSA ratio go higher up to 1:1:1:10, no significant influence in phosphopeptide fraction, although still 8 phosphopeptides were still detected, however, there are only 4 and 5 glycopeptides were detected in ratio 1:1:1:5 and 1:1:1:10 respectively.
Further application to the proteome level was demonstrated in PC9 cell line, a non-small cell lung cancer (NSCLC) cell line. By direct glycopeptide enrichment, 306 unique intact glycopeptides and 35 unique glycan structure were identified. With deglycosylation treatment by PNGase F, 1068 deamidated peptides with consensus motif NxS/T/V/C (x belongs to any amino acids but not proline) were detected. On the other hand, 246 phosphopeptides (231 mono-phosphorylated and 15 multi-phosphorylated) were identified in direct phosphopeptides. With initial 200μg membrane peptide from PC9 cell and followed by desalting and sequential enrichment strategy, 236 phosphopeptides (209 mono-phosphorylated and 27 multi-phosphorylated) were detected in phosphopeptide elute fraction. 243 unique intact glycopeptides (24 unique glycan structure) and 432 deamidated peptides with glycosylation motif (after deglycosylation) were identified in glycopeptide elute fraction combined with LC-MS/MS analysis using orbitrap fusion lumos mass spectrometry. These results further demonstrate that NH2/ZIC HILIC MNP can be a highly-potential method for characterization of both phosphopeptides and glycopeptides. | en_US |