Dr. Marco Fumagalli

LC-MS/MS for the identification and quantification of differentially expressed proteins in individuals with pulmonary emphysema associated to α1-antitrypsin deficiency

To prove the clinical efficacy of intravenous augmentation therapy for emphysema, caused by type ZZ deficiency of alpha-1-antitrypsin implies a need for biomarkers that serve as indicators of future clinical emphysema progression. With the advent of proteomics, the screening of biological fluids for the search of proteins as potential biomarkers of diseases has progressed remarkably. Given the non-invasive character of collection of Exhaled Breath Condensate (EBC), this fluid seems particularly attractive for monitoring alterations of the respiratory tract. In fact, provided that proteins in EBC likely reflect the composition of the airway-lining fluid, they can provide valuable information on lung disease state. The application of Liquid Chromatography-tandem Mass Spectrometry (LCMS/MS) to EBC of different cohorts of individuals has been previously reported. These proteomic analyses allowed to produce preliminary fingerprints of EBCs from non-smokers/healthy smokers and from COPD individuals with and without pulmonary emphysema associated with alpha-1-antitrypsin deficiency (AATD).

Aim:
Aim of the present study is to address the following questions:

  1. Do the EBC profiles that will be generated allow identification of proteins/combination of proteins that may be deregulated in the diseased compared to healthy status?
  2. Does the variation (over a period of time) in protein profile between sequential samples somehow reflect lung condition?
  3. In terms of molecular networks, which are the interaction partners of the proteins identified?

Methods

We previously collected EBC from 40 type ZZ AATD patients and 20 healthy partners at quarterly intervals under highly controlled circumstances over a 14 month period resulting in 5 EBC samples from each study participant.

We will use a very sophisticated Nano LC-Ultra 2D System combined with cHiPLC-nanoflex system in trap-elute mode and coupled to a QExactive mass spectrometer equipped with a nanospray ionization source to perform quantitative analyses of these EBC samples.

To understand whether differences in protein concentration of EBCs from patients and healthy controls are due to sample dilution only or are correlated to the physiological state of the lung, the dilution factor of each EBC will be exactly calculated by measuring the conductivity of each sample.

Objectives:

The main objective of this work is to generate protein profiles of EBCs useful for exploring protein-based pathological mechanisms in AATD.

A unique set of serial samples from healthy controls as partners of patients will help to identify the consistency of potential markers in AATD patients. Sample volumes are large enough to assess the reproducibility of indicative biomarkers in PiZZ patients with emphysema. Provided that differential quantitative proteomics can deliver a clear picture of molecular biological processes, the quantification of both the relative and absolute amounts of particular proteins in EBC is the critical goal of this project. The quantitative assessment of proteins identified will thus provide decisive progress for the identification of biomarkers of the disease.

Curriculum Vitae Dr. Marco Fumagalli:

Marco received his PhD in Bio-molecular Sciences and Biotechnology, at IUSS
Institute (University of Pavia). This experimental work enabled him to gain
experience in a variety of techniques including: extraction and purification of
proteins, methods for protein assay, mono- and two-dimensional electrophoresis,
capillary electrophoresis and liquid chromatography-mass spectrometry.
He is currently working (at the Department of Biology and Biotechnologies of the
University of Pavia) in the field of biological fluids proteomics.
His ALTA project "LC-MS/MS for the identification and quantification of
differentially expressed proteins in individuals with pulmonary emphysema
associated to α1-antitrypsin deficiency" focuses on generating protein profiles of
EBCs useful for exploring protein-based pathological mechanisms in AATD.

Contact:

Dr. Marco Fumagalli
Department of Biology and Biotechnologies
Biochemistry Unit
University of Pavia
Pavia, Italy