Archives

  • 2018-07
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • br STAR Methods br Introduction Glycosylation is one of

    2022-01-13


    STAR★Methods
    Introduction Glycosylation is one of the most prevalent post-translational modifications of proteins. It not only plays major roles in folding, transport and localization of proteins [1], but also regulates various biological processes such as cell growth, viral replication and immune defense [2], [3], [4]. Aberrant glycosylation is usually associated with the pathological progression of many diseases [5]. N-linked glycoproteins are widely distributed, ranging from surface of various types of 3-Chlorotyrosine to different human body fluids such as serum, cerebrospinal fluid and urine [5], [6]. Transmembrane or cell surface glycoproteins are easily accessible to therapeutic drugs, antibodies, and ligands while glycoproteins secreted in 3-Chlorotyrosine body fluids are thought to provide a detailed window into the state of health of an individual. These features make glycoproteins a highly interesting class of proteins for clinical and biological research. In the last decade, mass spectrometry (MS) has been widely used for large-scale glycoproteomic analysis. Typically, glycoproteins or glycopeptides are first enriched from complex samples using different enrichment methods, such as lectin affinity [7], hydrazide chemistry [8] and hydrophilic interaction liquid chromatography (HILIC) [9]; glycans are then cleaved from the glycoproteins or glycopeptides by enzymatic or chemical methods prior to MS analysis of deglycosylated peptides (Fig. 1). As for N-linked glycosylation, the deamidation which occurs at the formerly N-glycosylation sites during the PNGase F treatment can serve as a mass tag (+0.98 Da, or +3 Da when performed in H218O) for N-glycosylation site identification using mass spectrometry [7], [8]. This simple but effective strategy has been widely applied into the large-scale analysis of N-linked glycoproteomes in various samples from human as well as other species. Using this strategy, thousands of N-linked glycoproteins have been identified from various human-derived samples through identifying their glycosite-containing peptides using mass spectrometry [10]. Recently, we have collected more than 30,000 human glycosite-containing peptides, representing >10,000 N-glycosites from >7000 N-glycoproteins, from over 100 studies regarding to human glycosite-containing peptide analysis published since 2003. These human-derived samples include various body fluids, tissues and cell lines, which can be classified into more than 20 different sample type groups. The entire human glycoproteome database as well as sub-databases associated with individual body fluids or tissues can be found in the N-GlycositeAtlas website at nglycositeatlas.biomarkercenter.org. In this paper, we summarized the progress of MS-based identification of human N-linked glycoproteins, focusing mainly on the identification of N-glycoproteins and glycosylation sites based on the deglycosylated forms of the glycosite-containing peptides. The glycoproteomic data were classified into three major groups based on their sample sources, including human body fluids, tissues (and related cell lines), and some special cell types such as blood platelets, B-cells, T-cells, as well as spermatozoa. We also discussed the glycosylation at some atypical N-glycosylation sites identified previously by mass spectrometry. Despite the development of novel methods for intact glycopeptide analysis in recent years, these data are beyond the scope of this review.
    Glycoproteins in body liquids Body fluid is one of the main resources for the identification of disease-related biomarkers. Now that N-linked glycoproteins account for a large portion of the protein content in the body fluids, identifying the glycoprotein components in these body fluids is essential for their clinical utility. Till now, N-glycoproteomic data identified by MS has been reported in serum/plasma, urine, saliva, cerebrospinal fluid (CSF) and milk samples (Table 1).
    Glycoproteins in human tissues and related cell lines