Sensitive glycopeptide analysis by enrichment and LC-MS/MS

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Glycosylation plays an important role in many biological processes, and aberrant glycosylation has been found to play a critical role in many diseases and cancer progression. Studies of glycoproteins take two approaches for full characterization in that both the peptide backbones and glycan structures must be obtained during analysis. Changes in glycosylation during disease progression are key to discovering biomarkers for diagnosis. Glycoproteomic strategies for identification and quantification are monitored through liquid chromatography separation interfaced with tandem mass spectrometry in this work. Glycopeptides, however, are abundantly low and express poor ionization efficiencies. Enrichment techniques are needed to capture glycopeptides and remove interfering species. Three HILIC enrichment parameters were tested on cell line HTB-131 and compared to the optimized 5mg Cotton HILIC protocol for blood serum sample analysis. Tests were conducted with various cotton amounts, buffer solutions, and incubation times. For sufficient glycopeptide capturing and peptide removal, it was found that 5 mg of cotton, a washing buffer consisting of 90% ACN /0.1% FA, and one hour incubation time provided the best results. Under these conditions, 715 peptides were identified while 206 glycosylation sites were identified. Some sample loss was observed, but an approximate four times increase in signal was observed utilizing these enrichment conditions. In breast cancer metastasis to the brain the blood brain barrier, a region of the brain that regulates the entrance of ions, diseases, toxins, etc., fails to block breast cancer cells from crossing. Here, we present a study of identifying and quantifying the glycosylation of six breast and brain cancer cell lines using hydrophilic interaction liquid chromatography (HILIC) and electrostatic repulsion liquid chromatography (ERLIC) enrichments and LC-MS/MS analysis. Qualitative and quantitative analyses of N-linked glycosylation were performed by both enrichment techniques for individual and complementary comparison. Cancer glycopeptide biomarkers were identified and confirmed by chemometric and statistical evaluations. A total of 497 glycopeptides were characterized with 401 common glycopeptides (80.6% overlap) determined from both enrichment techniques. HILIC enrichment yielded 320 significant glycopeptides out of 494 unique glycopeptides, and sequential HILIC-ERLIC enrichment yielded 212 significant glycopeptides out of 404 unique glycopeptides. The results provide the first comprehensive glycopeptide listing for these six cell lines. Methods for surface analysis of biological samples include Secondary Ion Mass Spectrometry (SIMS), Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI MS), and Liquid Extraction Surface Analysis (LESA). LESA techniques were utilized in a previous study published from our lab. In this study on-tissue digestion was conducted for glycomic profiling of mouse brain tissue sections. Manual deposition of PNGase F enzyme was spotted on to regions of the tissue. A collection of the released glycans and analysis by LC-MS resulted in the identification of 43 glycan structures. The success of this experimental design led to the development of an automated instrument for enzyme deposition termed ‘Microliter Deposition Device.’ Different materials were tested for the creation of masks. These masks functioned as stencil-like templates for enzyme deposition by creating wells/holes of fixed diameters. Polyetheretherketone (PEEK), polyoxymethylene (POM; Delrin), and polytetrafluoroethylene (PTFE, Teflon) were the materials tested. It was found that the Teflon mask with the circular holes packed in a hexagonal pattern provided the most efficient sealing. The second generation microliter deposition device was then constructed to achieve automated on-tissue digestion. The new device had three step motors for the movement in the x, y, and z directions. It included fixed locations for the mask and well plate positioning. The device is controlled by a LabVIEW VI of 30 stacked sequences. The capabilities of this new device, include deposition and withdrawing control, syringe pump control, temperature control, increased precision, and increased spatial resolution. The goal of this project is to implement the device for on-tissue digestion and analysis eventually for imaging mass spectrometry (IMS).

N-glycan, Glycopeptide, HILIC enrichment, ERLIC enrichment, Imaging mass spectrometry, On-tissue digestion, Microliter deposition device