Mass Spectrometric Identification of Glycosylphosphatidylinositol-Anchored Peptides

Glycosylphosphatidylinositol (GPI) anchoring is a post-translational modification widely observed among eukaryotic membrane proteins. GPI anchors are attached to proteins via the carboxy-terminus in the outer leaflet of the cell membrane, where GPI-anchored proteins (GPI-APs) perform important functions as coreceptors and enzymes. Precursors of GPI-APs (Pre-GPI-APs) contain a C-terminal hydrophobic sequence that is involved in cleavage of the signal sequence from the protein and addition of the GPI anchor by the transamidase complex. In order to confirm that a given protein contains a GPI anchor, it is essential to identify the C-terminal peptide containing the GPI-anchor modification site (ω-site). Previously, efficient identification of GPI-anchored C-terminal peptides by mass spectrometry has been difficult, in part because of complex structure of the GPI-anchor moiety. We developed a method to experimentally identify GPI-APs and their ω-sites. In this method, a part of GPI-anchor moieties are removed from GPI-anchored peptides using phosphatidylinositol-specific phospholipase C (PI-PLC) and aqueous hydrogen fluoride (HF), and peptide sequence is then determined by mass spectrometry. Using this method, we successfully identified 10 GPI-APs and 12 ω-sites in the cultured ovarian adenocarcinoma cells, demonstrating that this method is useful for identifying efficiently GPI-APs

Mass Spectrometric Identification of Glycosylphosphatidylinositol-Anchored Peptides

Glycosylphosphatidylinositol (GPI) anchoring is a post-translational modification widely observed among eukaryotic membrane proteins. GPI anchors are attached to proteins via the carboxy-terminus in the outer leaflet of the cell membrane, where GPI-anchored proteins (GPI-APs) perform important functions as coreceptors and enzymes. Precursors of GPI-APs (Pre-GPI-APs) contain a C-terminal hydrophobic sequence that is involved in cleavage of the signal sequence from the protein and addition of the GPI anchor by the transamidase complex. In order to confirm that a given protein contains a GPI anchor, it is essential to identify the C-terminal peptide containing the GPI-anchor modification site (ω-site). Previously, efficient identification of GPI-anchored C-terminal peptides by mass spectrometry has been difficult, in part because of complex structure of the GPI-anchor moiety. We developed a method to experimentally identify GPI-APs and their ω-sites. In this method, a part of GPI-anchor moieties are removed from GPI-anchored peptides using phosphatidylinositol-specific phospholipase C (PI-PLC) and aqueous hydrogen fluoride (HF), and peptide sequence is then determined by mass spectrometry. Using this method, we successfully identified 10 GPI-APs and 12 ω-sites in the cultured ovarian adenocarcinoma cells, demonstrating that this method is useful for identifying efficiently GPI-APs

Clinical Significance of Tissue Factor Pathway Inhibitor 2, a Serum Biomarker Candidate for Ovarian Clear Cell Carcinoma - Fig 2

<p><b>ROC and AUC values for serum CA125 and TFPI2 levels in discrimination of CCC from other ovarian diseases (benign diseases, borderline, and non-CCC EOCs) (A), CCC versus borderline ovarian tumors (BD) and non-CCC EOCs (B), and CCC versus EMS (C).</b> Red line, TFPI2; black dotted line, CA125. Numbers indicate the calculated AUC values for TFPI2 and CA125.</p

Distribution of CA125 and TFPI2 in serum samples from patients with CCC and other gynecological diseases (retrospective training set, n = 268), along with TFPI2 mRNA levels in their tissue samples.

<p>Serum CA125 levels in patients with CCC, other ovarian diseases, and uterine (Ut) diseases (<b>A</b>). Distribution of CA125 levels at the time of sample collection. Serum TFPI2 levels in patients with CCC, other ovarian diseases, or Ut diseases in the training set (<b>B</b>). Serum TFPI2 levels in samples of the training set were measured by the Pre-Diluted Assay as described in Materials and Methods. Comparison of CA125 (<b>C</b>) and TFPI2 (<b>D</b>) levels in patients with EMS, non-EMS, each EOC subtypes, and various Ut tumors (UF: uterine fibroids, cervical cancer, endometrial cancer). Box plots display 5^th, 25^th, 50^th (median, middle horizontal line), 75^th, and 95^th, percentiles. Statistical analysis was performed by nonparametric Mann-Whitney <i>U</i> test.</p

Clinicopathologic characteristics of the patients.

<p>Clinicopathologic characteristics of the patients.</p

Sources of samples.

<p>Sources of samples.</p