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. 2012 Feb 4;10(1):7.
doi: 10.1186/1477-5956-10-7.

MS/MS library facilitated MRM quantification of native peptides prepared by denaturing ultrafiltration

Juraj Lenco  1 , Renny Lan, Nathan Edwards, Radoslav Goldman
Affiliations

MS/MS library facilitated MRM quantification of native peptides prepared by denaturing ultrafiltration

Juraj Lenco et al. Proteome Sci. .

Abstract

Naturally occurring native peptides provide important information about physiological states of an organism and its changes in disease conditions but protocols and methods for assessing their abundance are not well-developed. In this paper, we describe a simple procedure for the quantification of non-tryptic peptides in body fluids. The workflow includes an enrichment step followed by two-dimensional fractionation of native peptides and MS/MS data management facilitating the design and validation of LC- MRM MS assays. The added value of the workflow is demonstrated in the development of a triplex LC-MRM MS assay used for quantification of peptides potentially associated with the progression of liver disease to hepatocellular carcinoma.

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Figures

Figure 1
Figure 1
BCA (A) and HPLC-UV (B) quantification of peptides in the ultrafiltrates prepared in water, 20% Acetonitrile, or 6 M guanidine HCl. Each sample was prepared in triplicate.
Figure 2
Figure 2
Polypeptide concentration in the ultrafiltrates eluted with 35% and 80% AcN following C18-SPE desalting. Each sample was prepared in triplicate.
Figure 3
Figure 3
MALDI-TOF (A) and HPLC-UV (B) analysis of tryptic digests of BSA in water, 20% AcN, and 6 M guanidine HCl. The inverted spectra in panel A represent digests with trypsin compared to an appropriate solvent control. HPLC-UV chromatograms in panel B show peptide peaks after trypsin digestion in water and 20% AcN and the parent peak for undigested BSA protein in the presence of 6 M guanidine HCl. Each sample was prepared in triplicate and one representative spectrum or chromatogram is presented
Figure 4
Figure 4
Recovery of three peptides (FIBA, CO3. and CO4) from serum following 2 h and overnight incubations with the addition of 6 M guanidine HCl or water. Each sample was prepared in triplicate.
Figure 5
Figure 5
Intensity of fragment ions of the doubly charged FIBA peptide in the MS/MS spectrum; the most intense fragments from the QTOF spectrum correspond to the three most intense 4000 QTRAP MRM transitions.
Figure 6
Figure 6
Quantification of FIBA, CO3, and CO4 peptides in samples of healthy controls (C), chronic liver disease patients (LD), and hepatocellular cancer patients (HCC).
See this image and copyright information in PMC

References

    1. Li L, Sweedler JV. Peptides in the brain: mass spectrometry-based measurement approaches and challenges. Annu Rev Anal Chem (Palo Alto Calif) 2008;1:451–483. doi: 10.1146/annurev.anchem.1.031207.113053. - DOI - PubMed
    1. Mitchell JW, Atkins N Jr, Sweedler JV, Gillette MU. Direct cellular peptidomics of hypothalamic neurons. Front Neuroendocrinol. 2011;32:377–386. doi: 10.1016/j.yfrne.2011.02.005. - DOI - PMC - PubMed
    1. Thevis M, Bredehoft M, Kohler M, Schanzer W. Mass spectrometry-based analysis of IGF-1 and hGH. Handb Exp Pharmacol. 2010;195:201–207. - PubMed
    1. Gomes I, Dale CS, Casten K, Geigner MA, Gozzo FC, Ferro ES, Heimann AS, Devi LA. Hemoglobin-derived peptides as novel type of bioactive signaling molecules. AAPS J. 2010;12:658–669. doi: 10.1208/s12248-010-9217-x. - DOI - PMC - PubMed
    1. Keay SK, Szekely Z, Conrads TP, Veenstra TD, Barchi JJ Jr, Zhang CO, Koch KR, Michejda CJ. An antiproliferative factor from interstitial cystitis patients is a frizzled 8 protein-related sialoglycopeptide. Proc Natl Acad Sci USA. 2004;101:11803–11808. doi: 10.1073/pnas.0404509101. - DOI - PMC - PubMed