Sialic Acid-focused Quantitative Mouse Serum Glycoproteomics by Multiple Reaction Monitoring Assay

Abstract

Clinical proteomics focusing on the identification and validation of biomarkers and the discovery of proteins as therapeutic targets is an emerging and highly important area of proteomics. Biomarkers are measurable indicators of a specific biological state (particularly one relevant to the risk of contraction) and the presence or the stage of disease, and are thus expected to be useful for the prediction, detection, and diagnosis of disease as well as to follow the efficacy, toxicology, and side effects of drug treatment, and to provide new functional insights into biological processes.At present, proteomics methods based on mass spectrometry (MS) have emerged as the preferred strategy for discovery of diagnostic, prognostic, and therapeutic protein biomarkers. Most biomarker discovery studies use unbiased, "identified-based" approaches that rely on high performance mass spectrometers and extensive sample processing. Semiquantitative comparisons of protein relative abundance between disease and control patient samples are used to identify proteins that are differentially expressed and, thus, to populate lists of potential biomarkers. De novo proteomics discovery experiments often result in tens to hundreds of candidate biomarkers that must be subsequently verified in serum. However, despite the large numbers of putative biomarkers, only a small number of them are passed through the development and validation process into clinical practice, and their rate of introduction is declining. The first non-standard abbreviation (MS above is standard) must be footnoted the same as the abbreviation footnote, and MRM must be the first abbreviation in the list because it is the one footnoted. After that the order does not matter.Targeted proteomics using multiple reaction monitoring (MRM) 1 is emerging as a technology that complements the discovery capabilities of shotgun strategies as well as an alternative powerful novel MS-based approach to measure a series of candidate biomarkers (1-7). Therefore, MRM is expected to provide a powerful high throughput platform for biomarker validation, although clinical validation of novel biomarkers has been traditionally relying on immunoassays (8, 9). MRM exploits the unique capabilities of triple quadrupoles (QQQ) MS for quantitative analysis. In MRM, the first and the third quadrupoles act as filters to specifically select predefined m/z values corresponding to the peptide precursor ion and specific fragment ion of the peptide, whereas the second quadrupole serves as collision cell. Several such transitions (precursor/fragment ion pairs) are monitored over time, yielding a set of chromatographic traces with retention time and signal intensity for a specific transition as coordinates. These measurements have been multiplexed to provide 30 or From the

Terminal Galactosylation and Sialylation Switching on Membrane Glycoproteins upon TNF-Alpha-Induced Insulin Resistance in Adipocytes

“…Mapping the extreme complexity of the mammalian glycoproteome remains challenging, but recent advances in mass spectrometry have dramatically aided this endeavor. While large-scale glycopeptide identification is now feasible and hundreds of glycopeptides have been identified (27,(43)(44)(45)(46)(47)(48), systems-wide quantification of the glycoproteome has had little attention (49). The glycopeptide identification has been advanced by the development of specific glycopeptide search algorithms or modification of existing proteomics pipelines (50 -53).…”

Human Urinary Glycoproteomics; Attachment Site Specific Analysis of N- and O-Linked Glycosylations by CID and ECD

“…The N-glycans are typically removed by PNGase F treatment during these protocols and the site-specific information of N-glycan structures is usually not addressed. Only a few glycoproteomic studies, aimed at analyzing intact N-glycopeptides from biological samples, have been published (34,35). Also, by comparison to N-glycosylation, characterization of protein O-glycosylation is analytically more challenging for several reasons, e.g.…”

Altered Expression of Sialylated Glycoproteins in Breast Cancer Using Hydrazide Chemistry and Mass Spectrometry

“…Recently, modified SPEG method has been applied to the analysis of cell surface N-linked glycoproteins; however, the specificity to sialylated glycopeptides has not yet been determined (51,52). Kurogochi et al (46) reported a strategy for the analysis of sialic acid containing glycopeptides by specific oxidation of sialoglycopeptides and conjugation to hydrazide solid support. The conjugated sialoglycopeptides were released from the solid support by hydrolysis of hydrazone linkage between glycopeptides and hydrazide beads in acidic condition.…”

“…The released glycopeptides with regenerated aldehydes were labeled with 2-aminopyridine (2-AP) for MS analysis. The 2-APlabeled peptides could be further deglycosylated and analyzed by mass spectrometry (46). This method is based on the reversible hydrazone linkage for conjugation and release of sialoglycopeptides.…”