ANALYTICAL TECHNIQUES FOR BIOMOLECULES - MOD.2

The Analytical Techniques for Biomolecules course is part of the core educational activities of the Biomolecular Chemistry track within the Master's Degree Programme in Chemistry and Sustainable Technologies. The course is structured into two modules (referred to as Module 1 and Module 2), each comprising theoretical lectures (held in the classroom) and laboratory sessions. Both modules share the overarching goal of building broad knowledge and skills on the principles and applications of the most significant advanced techniques in the bioanalytical field, specifically:
- understanding the principles underlying bioanalytical techniques;
- developing the ability to propose, design, and manage new approaches in the bioanalytical field to solve complex problems related to the qualitative and quantitative determination of chemical species of interest in real matrices;
- acquiring skills to extract and communicate analytical information from experimental data, with full awareness of the capabilities, limitations, and uncertainties associated with techniques, application methods, analytes, matrices, and specific objectives;
- developing flexible and adaptive skills in the use of software tools for managing advanced instrumentation and processing chemical-analytical data, with a focus on automation, big data, and interoperability.

The expected learning outcomes are defined according to the specific content of the two modules that make up the Analytical Techniques for Biomolecules course.

The course aims to provide the knowledge and methodological foundations necessary for the development and application of advanced analytical procedures for the qualitative and quantitative determination of biomolecules in complex matrices.

At the end of the course, students will have acquired:
- knowledge of the role of bioanalytical chemistry in omics and post-omics sciences, with reference to targeted and untargeted analysis and to the main application contexts in the biological and biomedical fields;
- knowledge of the principles of experimental design, sampling, preservation, traceability, and sample preparation, as well as biosafety issues and the main ethical and legal implications;
- knowledge of the fundamentals of chromatographic separation in bioanalysis, with particular reference to gas chromatography and liquid chromatography;
- in-depth knowledge of the principles, instrumental configurations, acquisition modes, and main applications of mass spectrometry in bioanalysis, with particular attention to ionisation sources, mass analysers, spectrum interpretation, and hyphenated techniques.

Students will also be able to:
- rationally design a bioanalytical experimental approach according to the scientific question and the matrix under investigation;
- critically discuss the strengths, limitations, uncertainties, and fields of application of different analytical techniques;
- interpret chromatographic and mass spectrometric data, including mass spectra and results obtained by hyphenated techniques;
- relate targeted and untargeted analytical strategies to biomolecular research problems;
- communicate results, methodological choices, and interpretative limits in a correct and informed way using appropriate technical and scientific terminology.

Basic knowledge of analytical chemistry and statistics is required. The ability to understand technical and scientific texts in English is also useful, as it is necessary for following the lectures and discussing papers and case studies.

The course is devoted to the principles and applications of advanced analytical techniques for the study of biomolecules in complex matrices of biological, biochemical, and biomedical interest, with particular emphasis on mass spectrometry and hyphenated techniques. The course contents include an introduction to the role of bioanalytical chemistry in omics and post-omics sciences, with reference to genomics, transcriptomics, proteomics, metabolomics, metallomics, and exposomics; principles of experimental design in bioanalytical sciences, with particular attention to targeted and untargeted analysis, definition of experimental goals, comparison and control, replication, randomisation, stratification/blocking, and the management of confounding factors; biosafety issues, ethical and legal requirements, as well as criteria for sampling, preservation, traceability, and sample preparation for gaseous, liquid, and solid matrices.
The course also addresses the fundamentals of chromatographic separation in bioanalysis, with particular reference to gas chromatography and liquid chromatography, separation mechanisms, the main chromatographic performance parameters, derivatisation, sample-introduction modes, and the selection of columns and operating conditions, also in view of coupling with mass spettrometry detection.
The main core of the course is devoted to mass spectrometry in bioanalysis. In this context, the course covers the general principles of ion formation, transmission, and detection, the main features of mass spectra, the interpretation of molecular ions, fragments, and isotopic patterns, as well as the concepts of mass resolution and resolving power. The main ionisation sources used in GC-MS, LC-MS, MALDI-MS, and ICP-MS are examined in detail, with particular reference to electron ionisation (EI), chemical ionisation (CI), electrospray ionisation (ESI), atmospheric pressure chemical ionisation (APCI), atmospheric pressure photoionisation (APPI), matrix-assisted laser desorption/ionisation (MALDI), and inductively coupled plasma (ICP) sources. The course also covers the main low- and high-resolution mass analysers, with particular attention to time-of-flight, quadrupoles, traps, magnetic and electrostatic sectors, double-focusing instruments, and hybrid configurations, together with the main strategies for data acquisition and interpretation.
Particular attention is devoted to hyphenated techniques based on the coupling of chromatographic separation with mass spectrometry, to the related instrumental and methodological choices, and to their use in the qualitative and quantitative analysis of biomolecules, in targeted and untargeted strategies, in omics applications, sequencing, organic and inorganic bioimaging, and metallomics. Course contents are further explored through the critical discussion of case studies and application-oriented laboratory activities.

Required materials: slides and teaching materials presented and discussed during lectures and laboratory activities, made available by the lecturer through the course e-learning platform.
Optional materials/supplementary readings: scientific papers, case studies, and any additional bibliographic references that may be suggested by the lecturer during the course as further reading or complementary material.

Assessment is based on an oral examination. In the first part, students are asked to briefly and critically present a case study or scientific paper of their choice relevant to the course topics; this is followed by in-depth questions on the selected case study and then on other topics covered in the theoretical lectures and laboratory activities. The examination is aimed at assessing: knowledge of the principles of advanced bioanalytical techniques and pre-analytical methodologies; the ability to understand and critically discuss the strengths, limitations, and application fields of different analytical approaches; the ability to frame analytical problems in complex matrices; the ability to interpret analytical data and relate them to experimental questions; and the correct use of technical and scientific terminology.

The final mark of the Analytical Techniques for Biomolecules course is single, corresponding to the arithmetic average of the individual marks achieved in the two modules.

oral

The lecturer has a duty to ensure that the rules regarding the authenticity and originality of exam tests and papers are respected. Therefore, if there is suspicion of irregular conduct, an additional assessment may be conducted, which could differ from the original exam description.

Assessment Grid:
27-30: full mastery of the topics covered in the course, in particular of the principles and applications of mass spectrometry and hyphenated techniques in bioanalysis; ability to independently reorganise and critically apply the acquired knowledge to the discussion of complex analytical problems; ability to correctly interpret data and results and to use appropriate technical terminology.
23-26: fair to good knowledge of the topics covered in the course; fair ability to organise and apply information in the discussion of bioanalytical problems and case studies; adequate ability to interpret data and generally correct use of technical terminology.
18-22: basic or partial knowledge of the topics covered in the course; limited ability to apply knowledge to the discussion of bioanalytical techniques and problems; interpretation of data not always autonomous or fully correct; terminology not always used appropriately.
Honours will be awarded in the presence of excellent knowledge of the course contents, full ability to critically integrate principles, methods, data, and case studies, and a personal, rigorous, and original re-elaboration of the issues addressed.

The course is delivered through lectures, discussion of case studies from the scientific literature, and laboratory activities. Teaching activities are aimed at developing the understanding of the theoretical principles and application aspects of the main advanced bioanalytical techniques covered in the course, with particular emphasis on mass spectrometry and hyphenated techniques, as well as the ability to critically interpret data and results. Laboratory activities are application-oriented and allow further exploration of selected topics addressed during the course, including aspects related to the hardware and software management of instrumentation and data processing.