Dataset Information

SMD-IPTL 4-plex yeast and BSA-yeast

ABSTRACT: Isobaric peptide termini labeling (IPTL) is an attractive protein quantification method, because it provides more accurate and reliable quantification information than traditional isobaric labelling methods (TMT, iTRAQ) by making use of the entire fragment ion series instead of only a single reporter ion. The multiplexing capacity of published IPTL implementations is, however, limited to three. Here, we present a selective maleylation-directed isobaric peptide termini labelling (SMD-IPTL) approach for quantitative proteomics of LysC protein digests. SMD-IPTL extends the multiplexing capacity to 4-plex with the potential for higher levels of multiplexing using commercially available 13C/15N-labelled amino acids. SMD-IPTL is achieved in a one-pot reaction in three consecutive steps: 1) selective maleylation at the N-terminus; 2) labelling at the ԑ-NH2 group of the C-terminal Lys with isotopically labelled acetyl alanine; 3) thiol Michael addition of an isotopically labelled acetyl cysteine at the maleylated N-terminus. The isobarically labelled peptides are fragmented into sets of b- and y-ion clusters upon LC-MS/MS, which not only convey sequence information but also quantitative information for every labelling channel and avoid the issue of ratio distortion observed with reporter-ion-based approaches. We demonstrate the SMD-IPTL approach with a 4-plex labelled sample of BSA and yeast lysates mixed at different ratios. Utilizing SMD-IPTL for labelling and a narrow precursor isolation window of 0.8 Th with an offset of -0.2 Th, accurate ratios were measured across a 10-fold mixing range of BSA in a background of yeast proteome. With the yeast proteins mixed at ratios of 1:5:1:5, BSA was detected at ratios 0.94:2.46:4.70:9.92 when spiked at 1:2:5:10 ratios with an average standard deviation of peptide ratios of 0.34.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Xiaobo Tian

LAB HEAD: Rainer Bischoff

PROVIDER: PXD018589 | Pride | 2020-04-27

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	4-plex-Ac-Cys-Ma-Yeast-Ac-Ala-1-1-1-1.mgf	Mgf
	4-plex-Ac-Cys-Ma-Yeast-Ac-Ala-1-1-1-1.pride.mgf.gz	Mgf
	4-plex-Ac-Cys-Ma-Yeast-Ac-Ala-1-1-1-1.raw	Raw
	4-plex-Yeast-1-5-1-5-BSA-1-2-5-10.mgf	Mgf
	4-plex-Yeast-1-5-1-5-BSA-1-2-5-10.pride.mgf.gz	Mgf

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Publications

Selective Maleylation-Directed Isobaric Peptide Termini Labeling for Accurate Proteome Quantification.

Tian Xiaobo X de Vries Marcel P MP Visscher Susan W J SWJ Permentier Hjalmar P HP Bischoff Rainer R

Analytical chemistry 20200512 11

Isobaric peptide termini labeling (IPTL) is an attractive protein quantification method because it provides more accurate and reliable quantification information than traditional isobaric labeling methods (e.g., TMT and iTRAQ) by making use of the entire fragment-ion series instead of only a single reporter ion. The multiplexing capacity of published IPTL implementations is, however, limited to three. Here, we present a selective maleylation-directed isobaric peptide termini labeling (SMD-IPTL) ...[more]

PMID: 32319746

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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).