An Introduction to Mass Spectrometry Dr Alison E. Ashcroft, Mass Spectrometry Facility Manager, Astbury Centre for Structural Molecular Biology, Astbury Building , The University of Leeds. CONTENTS 1. What is mass spectrometry (MS)? What Information does mass spectrometry provide? 2. Where are mass spectrometers used? 3. How can mass spectrometry help biochemists? 4. How does a mass spectrometer work? 1. Introduction 2. Sample introduction 3. Methods of sample ionisation 4. Analysis and separation of sample ions 5. Detection and recording of sample ions 5. Electrospray ionisation 1. Electrospray ionisation 2. Nanospray ionisation 3. Data processing 6. Matrix assisted laser desorption ionisation 7. Positive or negative ionisation? 8. Tandem mass spectrometry (MS-MS): Structural and sequence information from mass spectrometry 1. Tandem mass spectrometry 2. Tandem mass spectrometry analyses 3. Peptide sequencing by tandem mass spectrometry 4. Oligonucleotide sequencing by tandem mass spectrometry 9. Background reading 1. What is mass spectrometry (MS)? What information does mass spectrometry provide? Mass spectrometry is an analytical tool used for measuring the molecular mass of a sample. For large samples such as biomolecules, molecular masses can be measured to within an accuracy of 0.01% of the total molecular mass of the sample i.e. within a 4 Daltons (Da) or atomic mass units (amu) error for a sample of 40,000 Da. This is sufficient to allow minor mass changes to be detected, e.g. the substitution of one amino acid for another, or a post-translational modification. For small organic molecules the molecular mass can be measured to within an accuracy of 5 ppm or less, which is often sufficient to confirm the molecular formula of a compound, and is also a standard requirement for publication in a chemical journal. Structural information can be generated using certain types of mass spectrometers, usually those with multiple analysers which are known as tandem mass spectrometers. This is achieved by fragmenting the sample inside the instrument and analysing the products generated. This procedure is useful for the structural elucidation of organic compounds and for peptide or oligonucleotide sequencing. 2. Where are mass spectrometers used? Mass spectrometers are used in industry and academia for both routine and research purposes. The following list is just a brief summary of the major mass spectrometric applications:?Biotechnology: the analysis of proteins, peptides, oligonucleotides ?Pharmaceutical: drug discovery, combinatorial chemistry, pharmacokinetics, drug metabolism ?Clinical: neonatal screening, haemoglobin analysis, drug testing ?Environmental: PAHs, PCBs, water quality, food contamination ?Geological: oil composition 3. How can mass spectrometry help biochemists? ?Accurate molecular weight measurements: sample confirmation, to determine the purity of a sample, to verify amino acid substitutions, to detect post-translational modifications, to calculate the number of disulphide bridges ?Reaction monitoring: to monitor enzyme reactions, chemical modification, protein digestion ?Amino acid sequencing: sequence confirmation, de novo characterisation of peptides, identification of proteins by database searching with a sequence “tag“ from a proteolytic fragment ?Oligonucleotide sequencing: the characterisation or quality control of oligonucleotides ?Protein structure: protein folding monitored by H/D exchange, protein-ligand complex formation under physiological conditions, macromolecular structure determination 4. How does a mass spectrometer work? 4.1 Introduction Mass spectrometers can be divided into three fundamental parts, namely the ionisation source , the analyser , and the detector. The sample has to be introduced into the ionisation source of the instrument. Once inside the ionisation source, the sample molecules are ionised, because ions are easier to manipulate than neutral molecules. These ions are extracted into the analyser region of the mass spectrometer where they are separated according to their mass (m) -to-charge (z) ratios (m/z) . The separated ions are detected and this signal sent to a data system where the m/z ratios are stored together with their relative abundance for presentation in the format of a m/z spectrum . The analyser and detector of the mass spectrometer, and often the ionisation source too, are maintained under high vacuum to give the ions a reasonable chance of travelling from one end of the instrument to the other without any hindrance from air molecules. The entire operation of the mass spectrometer, and often the sample introduction process also, is under complete data system control on modern mass spectrometers. Simplified schematic of a mass spectrometer 4.2 Sample introduction The method of sample introduction to the ionisation source often depends on the ionisation m