New proteins and protein species identified in human umbilical vein endothelial cells by Fourier transform ion cyclotron resonance-mass spectrometry

Bruno Baudin,Michel Vaubourdolle,Paulo Marcelo,Jean Rossier,Pascal Pernet,Arnaud Bruneel,Joëlle Vinh,Iman Haddad

doi:10.4236/ns.2013.58111

Bruno Baudin, Michel Vaubourdolle + Show 6 more

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https://doi.org/10.4236/ns.2013.58111

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Journal: Natural Science	Publication Date: Jan 1, 2013
Citations: 12	License type: CC BY 4.0

Abstract

For many years, HUVEC.com1 public database provides biological data relative to the proteome of human umbilical vein endothelial cells (HU-VECs), which are the most used human endothelial cell model in vascular biology. The proteins were identified using two-dimensional gel electrophoresis (2-DGE) for protein separation coupled with Matrix Assisted Laser Desorption-Ionization Mass Spectrometry (MALDI-TOF-MS) for identification. We present here an important update of HUVEC.com with 521 protein identifications as determined using Fourier transformed ion cyclotron resonance-mass spectrometry (FTICR-MS) applied to an unstained 2-DGE gel cut in 221 squared pieces; each identified protein being accompanied by a semi-quantitative three dimensional visualization is called “score imaging”. The squared analyzed gel and the alphabetical list of identified proteins, linked with their corresponding three-dimensional score imaging, are available at www.huvec.com. This original approach led to the establishment of the most protein-rich and informative database for HUVECs, as well as to the identification of some protein species, in particular with phosphorylation.

Highlights

From 2004, HUVEC.com shared a public database relative to human umbilical vein endothelial cells (HUVECs) proteome as assessed by the classical peptide mass fingerprinting approach combining two-dimensional gel electrophoresis (2-DGE) and Matrix Assisted Laser Desorption-Ionization Mass Spectrometry (MALDI-TOF-MS) [1]
In the goal to further enrich HUVEC.com, we used Fourier transformed ion cyclotron resonance-mass spectrometry (FTICR-MS) applied to an unstained 2. Two-Dimensional Gel Electrophoresis (2-DGE) gel cut in 221 equal rectangles to avoid the relatively poor sensitivity and the spot overlapping inherent to 2-DGE with classical staining [4,5]
Two identical gels were prepared: the first gel was stained successively with Colloidal Coomassie Blue (CCB) with silver nitrate; the second gel was cut in 221 equal rectangles without staining, the resulting grid pattern being matched against the stained control gel (Figure 1)

Summary

Introduction

From 2004, HUVEC.com (www.huvec.com) shared a public database relative to human umbilical vein endothelial cells (HUVECs) proteome as assessed by the classical peptide mass fingerprinting approach combining two-dimensional gel electrophoresis (2-DGE) and Matrix Assisted Laser Desorption-Ionization Mass Spectrometry (MALDI-TOF-MS) [1]. Encountering a good audience with more than 100,000 visits till September 2012, HUVEC.com database appears as notably insufficient especially because of being restricted to a relatively low number of major, mainly soluble endothelial proteins. In the goal to further enrich HUVEC.com, we used Fourier transformed ion cyclotron resonance-mass spectrometry (FTICR-MS) applied to an unstained 2-DGE gel cut in 221 equal rectangles to avoid the relatively poor sensitivity and the spot overlapping inherent to 2-DGE with classical staining [4,5]. This study allowed for identifying some protein species in HUVECs, such as heat-shock proteins and proteins from the cytoskeleton

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