The study of protein expression profiles for biomarker discovery in serum and in
mammalian cell populations needs the continuous improvement and combination of
proteins/peptides separation techniques, mass spectrometry, statistical and bioinformatic
approaches.
In this thesis work two different mass spectrometry-based protein profiling strategies have
been developed and applied to liver and inflammatory bowel diseases (IBDs) for the
discovery of new biomarkers.
The first of them, based on bulk solid-phase extraction combined with matrix-assisted laser
desorption/ionization - Time of Flight mass spectrometry (MALDI-TOF MS) and
chemometric analysis of serum samples, was applied to the study of serum protein
expression profiles both in IBDs (Crohn’s disease and ulcerative colitis) and in liver
diseases (cirrhosis, hepatocellular carcinoma, viral hepatitis).
The approach allowed the enrichment of serum proteins/peptides due to the high
interaction surface between analytes and solid phase and the high recovery due to the
elution step performed directly on the MALDI-target plate. Furthermore the use of
chemometric algorithm for the selection of the variables with higher discriminant power
permitted to evaluate patterns of 20-30 proteins involved in the differentiation and
classification of serum samples from healthy donors and diseased patients. These proteins
profiles permit to discriminate among the pathologies with an optimum classification and
prediction abilities. In particular in the study of inflammatory bowel diseases, after the
analysis using C18 of 129 serum samples from healthy donors and Crohn’s disease,
ulcerative colitis and inflammatory controls patients, a 90.7% of classification ability and a
72.9% prediction ability were obtained. In the study of liver diseases (hepatocellular
carcinoma, viral hepatitis and cirrhosis) a 80.6% of prediction ability was achieved using
IDA-Cu(II) as extraction procedure. The identification of the selected proteins by MALDITOF/
TOF MS analysis or by their selective enrichment followed by enzymatic digestion
and MS/MS analysis may give useful information in order to identify new biomarkers
involved in the diseases.
The second mass spectrometry-based protein profiling strategy developed was based on a
label-free liquid chromatography electrospray ionization quadrupole - time of flight
differential analysis approach (LC ESI-QTOF MS), combined with targeted MS/MS
analysis of only identified differences. The strategy was used for biomarker discovery in
IBDs, and in particular of Crohn’s disease. The enriched serum peptidome and the
subcellular fractions of intestinal epithelial cells (IECs) from healthy donors and Crohn’s
disease patients were analysed.
The combining of the low molecular weight serum proteins enrichment step and the LCMS
approach allowed to evaluate a pattern of peptides derived from specific exoprotease
activity in the coagulation and complement activation pathways. Among these peptides,
particularly interesting was the discovery of clusters of peptides from fibrinopeptide A,
Apolipoprotein E and A4, and complement C3 and C4. Further studies need to be
performed to evaluate the specificity of these clusters and validate the results, in order to
develop a rapid serum diagnostic test.
The analysis by label-free LC ESI-QTOF MS differential analysis of the subcellular
fractions of IECs from Crohn’s disease patients and healthy donors permitted to find many
proteins that could be involved in the inflammation process. Among them heat shock
protein 70, tryptase alpha-1 precursor and proteins whose upregulation can be explained
by the increased activity of IECs in Crohn’s disease were identified. Follow-up studies for
the validation of the results and the in-depth investigation of the inflammation pathways
involved in the disease will be performed.
Both the developed mass spectrometry-based protein profiling strategies have been
proved to be useful tools for the discovery of disease biomarkers that need to be validated
in further studies.
| Identifer | oai:union.ndltd.org:unibo.it/oai:amsdottorato.cib.unibo.it:1025 |
| Date | 18 March 2008 |
| Creators | Nanni, Paolo <1979> |
| Contributors | Roda, Aldo |
| Publisher | Alma Mater Studiorum - Università di Bologna |
| Source Sets | Università di Bologna |
| Language | English |
| Detected Language | English |
| Type | Doctoral Thesis, PeerReviewed |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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