Mass spectrometry (MS) is used in several fields, e.g. biology, environmental monitoring, medicine and forensics due to excellent qualitative and quantitative capabilities. The development of new instrumental setups and ionization sources is crucial to analyze a variety of compounds at trace levels. The synergy between material science and analytical chemistry allowed the development of new materials characterized by specific features of polarity, porosity and functionalization, able to interact with targeted analytes in complex matrices, resulting in high extraction efficiency even in presence of overwhelming amounts of interfering compounds. New methods based on the use of new materials and MS techniques for analytes extraction and detection have been proposed, providing fast analysis times, enhanced selectivity and increased sensitivity.

In this thesis, the development of new materials and setups for mass spectrometric applications is discussed.

In Paper I-III the design, synthesis, characterization and evaluation of the analytical performances of four new supramolecular receptors for targeted extraction of benzene, toluene, ethylbenzene and xylenes (BTEX) are reported. The synthesized materials were used as solid-phase microextraction coatings (SPME) for the GC-MS determination of BTEX at trace levels in urban air. In addition, a portable device for in-situ and real-time monitoring of BTEX using these receptors in the preconcentration unit is presented.

In Paper IV the development of coated ion sources able to improve the performances of an interface coupling liquid chromatography (LC) and electron ionization (EI), called Direct-EI LC-MS, is discussed. The coatings, obtained by sol-gel technique, were deposited onto commercial stainless steel EI sources to increase the inertness of its vaporization surface.

In Paper V, a rapid screening method for the detection of new psychoactive substances (NPS) in oral fluids is presented. New slides based on polylactide (PLLA), carbon particles and silica were tested as probe materials to promote the ionization of the analytes in desorption electrospray ionization – high resolution mass spectrometry (DESI-HRMS). Microextraction by packed sorbent (MEPS) of the analytes from the saliva samples was required due to the high signal suppression. The developed MEPS-DESI-HRMS method was validated and applied for the determination of NPS in road-collected samples.

In Paper VI the development of a new setup called solvent assisted paper spray ionization (SAPSI) is reported. This integrated solution allowed the increased data acquisition time and a close control over the ionization conditions. It was applied for the analysis of biomolecules, namely proteins, lipids, glycans, and amyloid peptides/aggregates, in aqueous solution as well as in human serum and cerebrospinal fluid. Different oligomeric species of amyloid aggregates were detected and it was possible to perform real-time monitoring of disaggregation processes. Modified protein species of physiological relevance such as oxidation, cysteinylation, glycosylation and glycation, and adduct formation were identified.

In conclusion, the new materials and setups discussed in this thesis allowed the development of selective and sensitive MS methods for the determination of different target compounds in complex matrices at trace levels with reduced sample pretreatment.

Glycosylation is a post-translational modification (PTM) that exerts profound structural and functional effects on the modified protein. Glycan synthesis and conjugation to proteins are regulated by a myriad of factors, both genetic and environmental, and are also influenced by external stressors. Glycosylation patterns are known to vary in correlation to a large number of diseases; therefore, it is possible to study such alterations to identify reliable biomarkers and help elucidate mechanisms underlying the disease. For these reasons, the development of analytical methods able to investigate the glycosylation of proteins in complex samples and to measure and characterize disease-related alterations is of great importance.

In this thesis, the development and application of rapid and small-scale methods for the analysis of the glycosylation pattern on specific proteins in biological fluids, with a high degree of automation and potential for parallel sample treatment, is presented.

Paper I illustrates a profiling method based on a microfluidic compact disc (CD) and its application to humans serum samples. The workflow integrated all the sample preparation steps, allowing a high degree of automation and sample treatment parallelization, significantly reducing the required processing time. In Paper II, a bead-based procedure for the immunoaffinity extraction of selected proteins from complex biological matrices was developed. This procedure improved and extended the applicability of the microfluidic CD method, increasing the flexibility and maintaining a good potential for automation. Paper III included a derivatization procedure in the bead-based methodology, to stabilize sialic acids for matrix-assisted lased desorption/ionization (MALDI) and to discriminate between connectivity isomers. Additionally, the method was applied to different biological fluids in order to highlight interpersonal variations of glycosylation. To increase the sample throughput, the method was scaled to a multi-wells format in Paper IV and subsequently applied to the investigation of alterations in the glycosylation pattern correlated to Alzheimer’s disease.

Papers V and VI focus on applications based on electrospray ionization (ESI). In Paper V, a source for paper spray ionization (PSI) was modified to create a new set-up to extend the applicability of this mass spectrometry (MS) technique to large biomolecules. It was possible to measure intact proteins, identifying many glycoforms together with other PTMs, as well as to characterize released glycans, performing structural analysis by tandem mass spectrometry (MS/MS). In Paper VI ESI-MS and the bead-based sample preparation method developed in Papers II, III, and IV were used for quantification of various glycoforms of intact proteins. Additionally, a travelling wave ion mobility spectrometry (TWIMS) MS/MS method was developed to structurally characterize the related N-glycans after enzymatic release.

The methods proposed in this thesis show valid approaches, which could be applied to investigate alterations of glycosylation at different levels, with potential implementation for biomarker investigation and development.