Dataset Information

Functions of peptides involved in cancer metastasis

ABSTRACT: We retrospectively investigated sera, obtained from 47 patients after the complete cure of liver cancer, using follow-up proteome analysis for 8 years, and found a protein, whose expression increased over time, peaked at the definitive diagnosis of bone metastases by bone scintigraphy, and tapered after the start of subsequent treatment. Fractions obtained by the reverse-phase HPLC of this protein were digested with trypsin and examined by LC-MS/MS analysis. The data were analyzed with a Mascot database search. As a result, the mass of fragment 576-628 of the C-terminal region of alpha-fibrinogen precursor was 5,805, which corresponded to that of the target peak. On the other hand, the mass of region 576-629 (with Val at the C-terminal) was 5,904, a mass value 99 higher than the target peak. This corresponds to a peak detected in the serum sample at a mass value 100 higher than the target peak. This peak with a mass value 100 higher was the same as the target peak in terms of chromatographic behavior, indicating that this peak had a protein sequence similar to that of the target peak. The isoelectric point (pI) of the fragment of region 576-628 was 8.07. This is consistent with the fact that the target peak indicates the behavior of a basic material in ion exchange. Thus, we concluded that the target peak 5,813 was a fragment of C-terminal region 576-628 of the alpha-fibrinogen precursor.

ORGANISM(S): Homo sapiens

PROVIDER: GSE43105 | GEO | 2017/12/16

SECONDARY ACCESSION(S): PRJNA184409

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:Abstract still has to be written. The obtained peptide mixtures were introduced into an LC-MS/MS system, the Ultimate 3000 (Dionex, Amsterdam, The Netherlands) in-line connected to an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Samples were first loaded on a trapping column (made in-house, 100 um internal diameter (I.D.) x 20 mm, 5 um beads C18 Reprosil-HD, Dr. Maisch). After back-flushing from the trapping column, the sample was loaded on a reverse-phase column (made in-house, 75 um I.D. x 150 mm, 5 um beads C18 Reprosil-HD, Dr. Maisch). Peptides were loaded with solvent A (0.1% trifluoroacetic acid, 2% acetonitrile), and were separated with a linear gradient from 2% solvent A' (0.05% formic acid) to 55% solvent B' (0.05% formic acid and 80% acetonitrile) at a flow rate of 300 nL/min followed by a wash reaching 100% solvent B'. The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS acquisition for the six most abundant peaks in a given MS spectrum. Full scan MS spectra were acquired in the Orbitrap at a target value of 1E6 with a resolution of 60,000. The six most intense ions were then isolated for fragmentation in the linear ion trap, with a dynamic exclusion of 60 s. Peptides were fragmented after filling the ion trap at a target value of 1E4 ion counts. From the MS/MS data in each LC run, Mascot Generic Files were created using the Mascot Distiller software (version 2.3.01, Matrix Science). While generating these peak lists, grouping of spectra was allowed with a maximum intermediate retention time of 30 s and a maximum intermediate scan count of 5 was used where possible. Grouping was done with 0.005 Da precursor tolerance. A peak list was only generated when the MS/MS spectrum contained more than 10 peaks. There was no de-isotoping and the relative signal to noise limit was set at 2. These peak lists were then searched with the Mascot search engine (Matrix Science) using the Mascot Daemon interface (version 2.3, Matrix Science). Spectra were searched against the human (H. sapiens) Swiss-Prot database. 13C2D3-acetylation of lysine side-chains, carbamidomethylation of cysteine and methionine oxidation to methionine-sulfoxide were set as fixed modifications for the N-terminal COFRADIC analyses. Variable modifications were 13C2D3-acetylation and acetylation of protein N-termini. Pyroglutamate formation of N-terminal glutamine was additionally set as a variable modification. Mass tolerance on precursor ions was set to 10 ppm (with Mascot's C13 option set to 1) and on fragment ions to 0.5 Da. Endoproteinase semi-Arg-C/P (Arg-C specificity with arginine-proline cleavage allowed) was set as enzyme allowing no missed cleavages. The peptide charge was set to 1+, 2+, 3+ and instrument setting was put to ESI-TRAP. Only peptides that were ranked one and scored above the threshold score, set at 99% confidence, were withheld. Quantification of the degree of Nt-Acetylation was performed as described previously (1). All data management was done in ms_lims (2).

Dataset Information

Functions of peptides involved in cancer metastasis

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets