Intact Protein Analysis (IPA)

Samenvatting

Proteins are large and complex macromolecules that play a critical role in all biological processes. In order to understand cellular mechanisms, scientists are analyzing the protein composition of cells, aiming to identify macromolecular actors and characterize their dynamics in physiological and pathological states.
State-of-the-art proteomics, using a combination of fractionation, digestion, liquid chromatography and mass-spectrometry, has enabled the identification of a large number of protein cellular components (> 4000 in complex organisms). However, the current approach reveals little on the distribution of proteins in proteoforms (isoforms and post-translational modification). Proteoforms are essential to characterize protein activities, their relationships and ultimately the cell status. The need to realize isoform-specific analysis has, therefore, led to new proteomics approaches.
The direct analysis of proteins has a great potential in proteoform analysis. However, the lack of efficient and automated separation methods compatible with mass spectrometry critically limits this approach. In the Intact Protein Analysis project I will develop and improve technologies to overcome current limitations, using novel combinations of advanced separation techniques and innovative chromatographic materials. The IPA approach is based on the online coupling of gel electrophoresis, two-dimensional liquid chromatography and mass spectrometry. The online nature of the proposed method will decrease the sample requirements and increase the analysis throughput, thanks to a reduction of the sample-handling steps, dilutions and losses. Moreover, the obtained information on the size, charge state and chemical characteristics of the analyzed species will be used in conjunction with mass-spectrometry data to increase the confidence in protein identification.
The development of such an orthogonal multidimensional analytical system will ensure high separation capacity, introducing a novel and powerful platform to characterize proteoforms present in complex cellular matrices and thus helping the development of applied proteomics (e.g. biomarker discovery, cancer research).

Producten

Wetenschappelijk artikel

  • A.F.G Gargano, A.B. Baglai(2017): Comprehensive lipidomic analysis of human plasma using multidimensional liquid- and gas-phase separations: Two-dimensional liquid chromatography–mass spectrometry vs. liquid chromatography–trapped-ion-mobility–mass spectrometry Journal of Chromatography A Volume 1530, 29 December 2017, Pages 90-103 pp. 90 - 103
  • P.J. Schoenmakers, A.F.G. Gargano, B.W.J. Pirok(2017): Optimizing separations in online comprehensive two-dimensional liquid chromatography Journal of Separation Science pp. 68 - 98

Kenmerken

Projectnummer

722.015.009

Hoofdaanvrager

Dr. A.F.G. Gargano

Verbonden aan

Universiteit van Amsterdam, Faculteit der Natuurwetenschappen, Wiskunde en Informatica, Van 't Hoff Institute for Molecular Sciences (HIMS)

Uitvoerders

Dr. A.F.G. Gargano

Looptijd

15/01/2016 tot 15/01/2019