Shotgun Proteomics Analysis of Estrogen Effects in the Uterus Using Two-Dimensional Liquid Chromatography and Tandem Mass Spectrometry

  • Eduardo A. Callegari
Part of the Methods in Molecular Biology book series (MIMB, volume 1366)


Shotgun (gel-free) proteomics is a useful approach to perform identification and relative quantification of protein in complex mixtures such as tissue homogenates, biological fluids, cell lysates, and extracellular proteins. Incorporation of separative and analytical techniques such as two-dimensional liquid chromatography at nanoscale (2D-nanoLC) coupled to tandem mass spectrometry (MS/MS analysis) into the shotgun protocol provides an excellent strategy. This chapter describes the application of the shotgun proteomics protocol to evaluate the identity and expression analysis of proteins from rat uterus after estrogen (ethinylestradiol) treatment. The steps of the protocol involve sample preparation (digestion), 2D-nanoLC-MS/MS analysis, and shotgun proteomics analysis including bioinformatics tools for data conversion, organization, and interpretation.

Key words

Proteomics “Shotgun” proteomics Liquid chromatography Mass spectrometry Proteins Peptides Rat Uterus Estrogen 



This work was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103443. The content is solely the responsibility of the author and does not necessarily represent the official views of the National Institutes of Health. The author would like to thank Dr. Kathleen Eyster for her generously providing the samples, and Dr. Barbara Goodman for her critical reading of the chapter. All sample analysis was performed in the SD BRIN Proteomics Core Facility. Mr. Douglas Jennewein, through the ITS Research Computing staff, contributed valuable technical expertise to this project.


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Division of Basic Biomedical Sciences, Sanford School of MedicineUniversity of South DakotaVermillionUSA

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