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Online Microreactor Titanium Dioxide RPLC-LTQ-Orbitrap MS Automated Platform for Shotgun Analysis of (Phospho) Proteins in Human Amniotic Fluid

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Abstract

Biomarkers in amniotic fluid (AF) include both non-modified and phosphorylated proteins and can be used in the diagnosis of pregnancy-associated pathologic conditions. In this work, an integrated LC–MS method for selective, sensitive and reproducible analysis of phosphorylation in proteins has been applied to AF. Online digestion of (phospho) proteins was coupled with the selective enrichment on a TiO2 trap, and separated by RPLC–MSn of both normal and phosphorylated produced peptides. First, an AF-pooled sample was analyzed and a general map of contained proteins and biomarkers was derived in a single run. Then, individual AF samples were analyzed with a downscaled platform with improved sensitivity. On purpose, a trypsin-based CIM® minidisk was used for online digestion of AF. The obtained protein profile was highly consistent with the one obtained with traditional off-line digestions. Moreover, the use of a specific phospho-enrichment tool followed by LTQ-Orbitrap, enhanced the confidence in the determination of protein phosphorylation state and phosphorylation sites. The phosphorylation sites of IGFBP-1 and osteopontin present in the AF of two individual samples were monitored with a total of 24 and 17 phosphopeptides, respectively, encoding for 12 putative novel phosphorylation sites in addition to known sites.

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Acknowledgments

The authors are grateful to Dr. Federica Corana, from Centro Grandi Strumenti, University of Pavia, for advice on LTQ instrument. This work was supported by PRIN grant 2007.

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Authors and Affiliations

  1. Department of Drug Sciences, University of Pavia, via Taramelli 12, 27100, Pavia, Italy

    Caterina Temporini, Enrica Calleri & Gabriella Massolini

  2. Laboratory of Analytical Pharmaceutical Chemistry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland

    Raul Nicoli, Serge Rudaz & Jean-Luc Veuthey

  3. Department of Computer Engineering and Systems Science of Pavia, University of Pavia, Via Ferrata 1, 27100, Pavia, Italy

    Alessandra Tiengo, Nicola Barbarini & Paolo Magni

  4. Department of Biochemistry, University of Pavia, viale Taramelli 3, 27100, Pavia, Italy

    Monica Galliano

  5. Department of Pharmaceutical Sciences “Pietro Pratesi”, University of Milan, via Mangiagalli 25, 20133, Milan, Italy

    Luca Regazzoni & Giancarlo Aldini

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  1. Caterina Temporini
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  2. Raul Nicoli
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  3. Alessandra Tiengo
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Correspondence to Caterina Temporini.

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Supplementary Information Available

Supplementary Materials and Methods and Figures (1–3) are available in Online Resource 1. Supplementary Table 1 and Table 2 are available in Online Resource 1; Supplementary Table 3 and Table 4 are available in Online Resource 2; Database A and Database B are available free of charge.

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Temporini, C., Nicoli, R., Tiengo, A. et al. Online Microreactor Titanium Dioxide RPLC-LTQ-Orbitrap MS Automated Platform for Shotgun Analysis of (Phospho) Proteins in Human Amniotic Fluid. Chromatographia 77, 39–50 (2014). https://doi.org/10.1007/s10337-013-2567-7

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