Ricerc@Sapienza

Native MS is a non-denaturing technique which allows to preserve noncovalent interactions and quaternary protein structure in the gas phase. Unlike conventional denaturing MS, which often includes organic solvents and low pH, native MS uses volatile, aqueous buffers at near physiological pH and gentler temperatures and voltages. This enables the study of protein-protein interactions, protein-ligand interactions, subunit architecture, and complex stoichiometry.
Native MS is compatible with a wide mass range (from small molecules to large multiprotein complexes) and requires only few microliters of sample at high nM to low uM concentration. This is a label free technique and prevents potential issues related to labelling or immobilization which may alter the intrinsic properties of proteins or their interactions.
The Q Exactive UHMR represents the latest advance in native MS technology allowing efficient transmission of large and heterogeneous protein complexes and resolution of small mass differences. The resolution is substantially better than other instruments, and its sensitivity is orders of magnitude higher. Compared to Time-of-Flight (ToF)-based instruments, the Q Exactive has incomparable transmission and sensitivity and, thanks to the latest advancements in the Orbitrap technology, it bridged mass range gap.
Given its extended mass range, it allows the transmission and analysis of complexes up to MegaDalton size.
At present, another Q Exactive platform is present in Sapienza (Dept. of Chemistry), however that instrument is a Q Exactive "classic" and not Ultra-High Mass Range (UHMR), is devoid of extended mass range and is designed for small molecule and peptide analysis. The Q Exactive "classic" cannot be upgraded to a UHMR because the two instruments present i) large hardware differences in all regions, ii) differences in quadrupole, which is vital for transmission of large, intact macromolecules, iii) new in-source trapping technology in the source region of the UHMR, which aids desolvation of large ions and is a key factor to successfully observe macromolecular complexes and perform native top-down MS. A MALDI-ToF/ToF instrument is present in the Dept. of Chemistry and Technologies of Drugs (CTD), but is mainly devoted to protein quality control, peptide/protein sequencing, mass spectrometry imaging. From an inspection of the instruments currently available in the Sapienza Research Infrastructure (SRI), we concluded that none of the currently available mass spectrometers has the right features to observe intact proteins and to preserve non-covalent interactions. A summary of the SRI mass spectrometers and their limitations is given in Table 1 (see previous box).
The Q Exactive UHMR will enable unprecedented research in the field of structural biology, allowing great integration with the current research lines of Sapienza, such as drug discovery for oncology and infectious diseases (Dept. CTD) or structure-based investigation of human pathologies such as cancer and neurodegeneration [Depts. of Biochemical Sciences (BS) and Biology and Biotechnologies (BB)].
The Q Exactive UHMR would be the only instrument with such potential not only in Sapienza, but all over Italy, thereby attracting national and international collaborators. It will allow design and contribution to high-impact projects aimed at the investigation of protein interactions, target identification and validation, and protein dynamics clarification. Moreover, from a more technical point of view, it will aid structural biology research on a daily basis, providing key information on endogenous ligand binding and subcomplex composition.
Our vision is to establish a native MS platform in Italy. Part of this vision includes the instrumentation itself, but a key part is also establishing collaborations with researchers in Italy and abroad. We see significant opportunity to train emerging researchers in this cutting-edge technique, thanking to the expertise acquired by our post-doc Dr. Fiorentino, who obtained his PhD in Oxford under the guide of C. Robinson, a pioneer of this technology. Native MS is frequently a key technique in the investigations of researchers from many different fields. This naturally means that there will be a broad contingent of italian and international researchers, from many different institutions, involved with a diversity of multidisciplinary science within INCENTIVE MS.
In the long term, along with collaborations, the Q Exactive UHMR will attract increasing funding opportunities. The interdisciplinary potential of this mass spectrometer will be pivotal for obtaining national and European funds and, given the absence of such an instrument in Italy, it will attract collaborations from academia and biotech/pharma, leading to technology tranfer and funding opportunities.