Mass Spectrometry Platform: Proteomics and Metabolomics
Leader: Prof Fulvio Magni
Mass Spectrometry Platform dedicated mainly to Proteomics and Metabolomics

A team of chemists, biochemists, clinical biochemists and clinicians operating out a modern platform located in the "House of Research and Innovation" (U28 Building). This multi-disciplinary team focuses on the characterisation of molecular alterations related to the bio / physiological processes which are at the root of various human pathologies. This includes the validation of markers as well as the identification and quantification of small molecules in different types of samples. The team has active collaborations with both public and private bodies to which it offers its expertise in various sectors (pharmaceutical, food, cosmetic, environmental and clinical) at both a national and international level. It is able to develop and validate analytical methods based on mass spectrometry.

Available technological resources and expertise
Expertise
The team has consolidated its experience in various application sectors including the analysis, by GC / LC-MS / MS, of drugs and small molecules as well as the study of the proteome and metabolome by mass spectrometry. In particular, in the latter field, high-throughput approaches, both top-down and bottom-up, can support projects aimed at identifying the proteome / peptidome, its qualitative alterations (post-translational modifications such as phosphorylation, N- glycosylation etc.) and quantitative in various types of samples, as well as validating the results. The team also has vast expertise in "Imaging" by mass spectrometry to obtain the spatial distribution and relative intensity of the molecules of interest directly in tissues and cells. The unit is also able to study the metabolome through untargeted and targeted identification / quantification techniques with both GC-MS / MS and with LC-MS / MS. Furthermore, the team has proven expertise in the management of different biological matrices for numerous biochemical studies, including those in which multiple centres are involved.
Specifically, the Proteomics and Metabolomics unit principally uses the following proteomics and metabolomics strategies for applications to liquid samples such as biological fluids (serum, urine, plasma, FNAB, etc.) and solids (tissues, cell cultures, microorganisms, regenerative scaffolds, etc.):

Proteomics
a) Identification of the proteome / peptidome and characterization of their modifications (eg phosphorylation, glycosylation, etc.) using shotgun and top-down approaches with nanoLC-ESI-QqTOF and nanoLC-ESI-ETD-MS / MS.
b) Relative quantification of peptides / proteins with both label-free and label-based protocols, by single and two-dimensional liquid chromatography combined with MS / MS.
c) Proteome / peptidome profile and in situ identification by MALDI-TOF / TOF.

IMAGING

d) Imaging by mass spectrometry to define the spatial localisation of intact proteins / peptides, N-glycans, lipids and small molecules within entire tissue sections and tissue microarrays (fresh frozen, FFPE) in order to build a molecular map of the sample.

Metabolomics

It is able to characterise the untargeted and targeted identification / quantification of the metabolome with either GC-MS / MS or LC-MS / MS.

Technological resources
The Mass Spectrometry platform has 2 MALDI-TOF / TOF, 2 LC-MS / MS and 1 GC-MS instrument. In addition, various instruments are also available for mono / bidimensional electrophoresis (2D-EF) and for antibody validation. The platform also has specific servers and software for managing mass spectrometry data and specific applications.

Instrumentation:
MALDI-TOF / TOF: rapiflex™ MALDI Tissuetyper™ and ultrafleXtreme™ (Bruker Daltonics);

These new generation mass spectrometers are of high accuracy and sensitivity. The two MALDI-TOF / TOF have a resolution of over 40,000 RP and a mass accuracy of better than 2-5ppm. The sensitivity is in the order of a few femtomoles.

The rapiflex™ MALDI Tissuetyper™ is specifically designed for MS imaging with a spatial resolution of 10-20 µm, so as to obtain information at the single cell level, and with a speed 10 times better than other commercial MS-Imaging tools.

NanoLC-ESI-MS / MS (Bruker Daltonics): nanoLC Ultimate 3000 interfaced with ESI-ETD-MS / MS (Amazon-ETD) and nanoEASY-LC combined with Impact Qq-TOF;
The nanoLC-ESI-Qq-TOF has a constant resolution of over 40,000 RP and an accuracy of up to 1-2 ppm. These two tools enable the rapid and reliable identification of proteins / peptides, their relative abundance and characterisation of their PTMs.

Data processing
The identity of proteins and peptides is obtained through Mascot (MatrixScience; in-house version) and Peaks Studio (Bioinformatics). The relative abundance of peptides and proteins in the samples, measured as a ratio, is calculated with Progenesis-QI (Waters) and Peaks Studio.
MS-Imaging data is processed with SCILS (vers. 2019c Pro) and with programs developed internally for specific purposes.

Contacts
fulvio.magni@unimib.it

02-64488213

clizia.chinello@unimib.it

02-64488105