Mass spectrometry Parts 1 and 2 -Adv Biochem -2022-23.pdf

Aims : To discuss the basic principles of mass spectrometry Outcomes : By the end of this lecture you should be able to describe the basis of mass spectrometry and its potential applications in scientific research. Part 1: General principles of mass spectrometry Dr Alan J Hargreaves Interd...

Aims : To discuss the basic principles of mass spectrometry Outcomes : By the end of this lecture you should be able to describe the basis of mass spectrometry and its potential applications in scientific research. Part 1: General principles of mass spectrometry Dr Alan J Hargreaves Interdisciplinary Biomedical Research Centre Ext n. 88061 Room 116 [email protected] It accurately measures the mass of a wide range of molecules from large (e.g. proteins) to small (e.g. sugars) What does a mass spectrometer do? Specifically : It identifies molecules in a mixture It detects impurities in a sample It can be used to analyse a purified protein or study the protein content of a sample from cells or tissues Research disciplines that use mass spectrometry Pharmaceutical industry: Drug discovery and development (e.g. metabolite identification and purity assessment of new products) Biochemistry research: Protein, peptide and oligonucleotide analysis, protein adduct formation, enzyme reactions, metabolites, etc. Clinical chemistry: Drug testing and neonatal screening. Food safety and environmental research. Forensic science Key components of a mass spectrometer • 3 components operating under vacuum: Detector: Records impact of individual ions high voltage acceleration Mass analyser: Separates ions according to mass/charge (m/z) ratios Source: Ionisation chamber Analyte converted to ions in vacuo Main stages of mass spectrometry 1) IONISATION : To be able to measure the mass of a molecule, a mass spectrometer converts it to a gas -phase ion. Ionisation occurs in one of 3 ways : Laser irradiation Electron ionisation Electrospray ionisation The resultant flux of electrically charged ions is converted into a proportional electrical current that can be read by the MS data analysis system to produce a mass spectrum Sources of samples analysed by mass spectrometry Clinical samples, cell and tissue extracts, environmental samples, etc. Instruments interfacing with MS : Gas liquid chromatography (GC/MS): environmental analysis, food safety screening, metabolomics, and clinical applications like forensics, toxicology, and drug screening. Liquid chromatography (LC/MS): Proteins and other small molecules not suitable for GC/MS Other sources of materials : SDS -PAGE or 2D -PAGE, purified proteins or other products Protein chip purification Nucleic acids and oligonucleotides, etc….. 2) ION SORTING This involves 2 steps Acceleration: + ve ions accelerate towards negative plates with speed depending on mass. Deflection: Ions are deflected in a magnetic field, again dependent on mass. Thus ions of different mass travel at different speeds and undergo different levels of deflection. 3) ION DETECTION Data from arrival times of different ions at the detector are converted into a mass spectrum. https://www.youtube.com/watch?v=NuIH9 -6Fm6U Simplified mass spectrum of pentane (CH 3CH 2CH 2CH 2CH 3) Peak heights of 2% or less of the base peak (the tallest peak) have been omitted Source: Spectral Data Base System for Organic Compounds (SDBS) at the National Institute of Materials and Chemical Research (Japan). Isotopic peaks Simple ions from small molecules tend to form a single ion giving a mono isotopic peak More complex molecules (e.g. peptide) produce multiple isotopic peaks http://www.waters.com/waters/en_GB/MS --- Mass -Spectrometry/nav.htm?cid=10073244&locale=en_GB M/Z ratio Relative abundance (%) Characteristic peaks produced in mass spectra • Each related to a specific mass ….. ….. …. ….. …. ….. ….. ….. ….. ….. ….. ….. nozzle sampling cone 1) Analyte dissolved & forced through narrow needle at high voltage 2) Fine spray of charged Droplets project from needle tip into vacuum chamber through nozzle 3) Acceleration through analyzer towards detector, as droplets dried in stream of inert gas Spray needle Electrospray After formation, the ions are “dragged” through a potential gradient (an electric field) to the counter plate. An electrospray mass spectrometer Other approaches Atmospheric Pressure Chemical Ionization (APCI) For samples unsuitable for gas phase ion conversion by ESI Involves transferring neutral analytes into the gas phase by vapourising the introduced liquid in a heated gas stream. Matrix assisted laser desorption ionisation Sample in matrix Laser Large molecular ions Mass determination MALDI - TOF mass spectrometers Summary of part 1 Mass spectrometry provides a measure of mass/charge ratio of ionised molecules It has an accuracy of molecular weight determination of 0.01 % Major applications in all areas of bioscience research http://www.youtube.com/watch?v=J -wao0O0_qM INTERVAL Part 2: Analysis of proteins and peptides by mass spectrometry Dr Alan J Hargreaves IBRC116 Tel ext n. 88061 [email protected] Aims (part 2) : To discuss the application of mass spectrometry and other proteomic techniques in the analysis of proteins and protein fragments Outcomes : By the end of this part of the lecture you should be able to discuss the main steps involved in the identification and sequencing of proteins using mass spectrometry Breaking protein into peptides and peptides into fragment Ions • Proteases, e.g., trypsin, break protein(s) into peptides which can be ionized and analysed by mass spectrometry (MS) • MS measures the mass to charge (m/z) ratio of each peptide ion • Tandem mass spectrometry (MS/MS) breaks the peptides down into fragment ions and measures the mass of each piece Types of sample Cell and tissue lysates/homogenates Subcellular fractions (e.g. mitochondria, nuclei, etc.) Partially purified proteins (e.g. chromatography) Proteins purified to homogeneity Etc. These samples may be analysed directly following trypsinization of: (a) samples in solution (b) protein bands separated by gel electrophoresis. Types of denaturing gel separation (a) Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS -PAGE) (b) Isoelectric focussing (IEF) (c) 2D -PAGE Typical stained gel after SDS - PAGE separation of porcine brain cytosol Stain used: Coomassie Brilliant Blue More sensitive alternatives: Silver stain reagent or fluorescent protein binding dyes (e.g. Sypro Ruby) (-) (+) Isoelectric focussing(IEF) Principles : Proteins separated by electrophoresis according to their net charge Sample buffer : Detergent (e.g. CHAPS), reducing agent (e.g. dithiothreitol), denaturing agent (e.g. 8M urea) - No SDS Gel Matrix : Polyacrylamide rod gels or Immobilised pH gradient (IPG) strips Main steps in 2D - PAGE 1) Proteins are separated according to net charge by electrophoresis in a pH gradient on IPG strips 2) Focussed samples on IPG strips are equilibrated with SDS - PAGE sample buffer in a 2 -step process involving reduction and carboxymethylation or carbamidomethylation of cysteine residues. 2) Bands are resolved by size in second dimension (as in SDS -PAGE), fixed and stained Separation of proteins by IEF and 2D - PAGE (a) and (b): Schematic diagrams of stained IPG strip and 2D gel, respectively. (c) and (d): Silver stained gel of neuroblastoma cell lysate (c) and Coomassie blue stained gel of same extract resolved by SDS - PAGE only (d). Spot Handling Workstation Spot Picker Digester Spotter Protein identification using a – 2D -PAGE -MS platform IEF SDS -PAGE scanned image analysis MALDI -TOF mass spectrometry Mass spectrometry and identification of proteins separated by gel electrophoresis via peptide mass fingerprinting ( pmf )Theoretical gene product: amino acid sequence Theoretical proteolytic peptides Match ? ! “ ” translationIn silico “ ” digestionIn silico DIPGHGQEVLIRLFKGHPETLEKFDKFKHLK SEDEMKASEDLKKHGATVLTALGGILKKKGH HEAEIKPLAQSHATKHKIPVKYLEFISECII VLQS DIPGHGQEVLIR LFKGHPETLEK FDKFKHLK SEDEMK ASEDLK “ ” digestion elution of peptides In vitro Peptide mass spectrum 2-D gel spot cutting Practical Experiment Genomic Database Search Genomic database: DNA Sequence Possible issues in pmf data • Post translational modifications such as: • Oxidation • Acetylation • Phosphorylation • Carboxymethylation • ADP ribosylation • Etc……. Further fragmentation of peaks: • E.g. by collisionally -induced decomposition • Laser + collisional gases (helium, argon, N 2 ) • Some instruments allow increased laser power • Analysis of ion fragments by “post source decay” (PSD) Peptide fragmentation Amino acids differ in their side chains Predominant fragmentation Weakest bonds Tendency of peptides to fragment at Asp (D) Mass Spectrometry in Proteomics Ruedi Aebersold* and David R. Goodlett 269 Chem. Rev. 2001, 101, 269 -295 C -terminal side of Asp CASE STUDY: Identification of novel biomarker of organophosphate toxicity in differentiating neuronal cells Harris et al (2009) Toxicology and Applied Pharmacology 240, 159 - 165 Galectin -1 α-enolase Actin a b PPCSI Cofilin a b Tubulin UCTH MIF GRP 78 kDa 150 - 100 - 75 - 50 - 37 - 25 - 15 - 3 pH gradient 10 CON DIAZ 3 10 3 10 kDa 150 - 100 - 75 - 50 - 37 - 25 - 15 - Image analysis using SameSpots Progenesis (Nonlinear) software, highlighting spots showing >2 -fold change Table 1 : Densitometric analysis and identification of protein of interest from 2 D-PAGE . __________________________________________________ __________________________________________________________ Protein group Estimated molecula r Mascot score Relative expression weight and pI (kDa/pH) (± sem ) __________________________________________________ __________________________________________________________ Cytoskeletal proteins : Actin 45 / 5.5 140 NSE Tubulin 52 / 5.0 74 NSE Cofilin - a 18 / 8.4 78 4.86 ± 1.9 3 Cofilin - b 4.65 ± 0.60 Cytosolic fa ctors: Galectin -1 10 / 4.9 53 1.7 2 ± 0.0 4 Macrophage migration inhibitory factor (MIF) 8 / 7.3 43 1.5 1 ± 0.06 -enolase 50 / 6.3 166 1.51 ± 0.15 Chaperone /proteasome proteins : Ubiqu itin C -terminal h ydrolase (UCTH) 24 / 5.3 50 .................... NSE Peptidyl -prolyl cis -trans isomerase (PPCTI -a) 16 / 7.4 112 112 1.67 ± 0.09 Peptidyl -prolyl cis -trans isomerase (PPCTI -b) 16 / 8.0 162 ................... 2.0 5 ± 0.5 1 Glucose regulated protein -78 (GRP -78) 77 / 5.1 219 ...... 1.7 3 ± 0.4 5 __________________________________________________ __________________________________________________________________ Validation of changes detected by MALDI -TOF MS Using a second approach (e.g. Western blotting) Effects of diazinon on cofilin level (% control value) 391 ± 61.2 p<0.01 183 ± 21.7 p<0.02 Summary: Part 2 Mass spectrometry and peptide mass fingerprinting of proteins separated by gel electrophoresis or liquid chromatography is a very effective way of identifying proteins in cell and tissue extracts In combination with appropriate image analysis software it can be used to identify protein changes under specified conditions. Good practice: Validation by alternative approaches in follow -up experiments.