RANIA MOHAMED EL-SHARKAWY Lecturer of clinical chemistry Medical Research Institute, Alexandria University MEDICAL RESEARCH INSTITUTE– ALEXANDRIA UNIVERSITY 15 th conference (3 rd international) ADVANCES IN MEDICAL RESEARCH ` From molecular medicine-to clinical application ` Mass spectrometric techniques

Mass Spectrometry

Mass spectrometry (MS) Mass spectrometry is a powerful analytical technique that is used to identify unknown compounds, to quantify known materials, and to elucidate the structure and chemical properties of molecules A mass spectrometer A mass spectrometer is a device that measures the mass-to-charge (m/z) ratio of ions.

Mass spectrometry

I. Sample Ionization

Simple Definition: Ionization is a process of charging a molecule. Molecules must be charged in order to measure them using a mass spectrometer

Types Types Of ionizing radiation Important to determine the target from analyte investigated

Hard ionization: the resulting unique fragmentation patterns can be used as a fingerprint for the identification of the sample. Fragment ions also provide important information about the primary structure (i.e. sequence) of the sample molecules. Soft ionization is applied to a stable molecule, the exact m/z value of its stable molecular thus, allows for the compositional analysis of the sample of unknown structure under study.

Mass spectrometry

A proton is transferred to or abstracted from,a gas phase analyte by a reagent gas molecule Chemical ionization Uses: analyte molecular mass determination & quantification  Little or no fragmentation as the protonated molecule is not highly excited in the chemical ionization

2-Electrospray Ionization (ESI)

Electro spray ionization

The 2002 Nobel Prize in Chemistry was awarded to two mass spectrometrists (J. Fenn and K. Tanaka) for their development of ionization techniques, which include electrospray ionization

3-Matrix Assisted Laser Desorption Ionization (MALDI)

SELDI -Modification of MALDI surface a type of affinity capture property -Sample of interest is exposed to affinity surface, certain analyte will preferentially bind -Washing to remove excess -Matrix is added to asset ionization Uses: analysis of protein mixture with low sample size (purification&analysis occur on the same surface

II. Analysis and Separation of Sample Ions

The mass analyzer separates the ions formed in the ionization source according to their mass-to-charge (m/z) ratios using some physical property e.g. electric or magnetic fields.

Time Of Flight Mass Analyzer (TOF)

Quadrupole Mass Analyzer

III. Detection of sample ions.

A tiny current is produced when the ion reaches the detector. The detector amplifies the signals which are then transmitted to the data system to be represented as peaks on a mass spectrum Mass spectrum: graphic representation of ions separated according to their m/z ratio

m/z Mass Spectrum

Mass spectrum of CO 2

Mass spectrum Questions needs to be asked?? 1.Negative or positive mode 2.Types of ion source result in different arrays of fragments produced from the original molecules. 3.origin of a sample 4. How the sample was prepared ??

Interpretation of mass spectra compare its experimental mass spectrum against a library of mass spectra compare its experimental mass spectrum against a library of mass spectra If the search comes up empty, then manual interpretation or software assisted interpretation of mass spectra are performed If the search comes up empty, then manual interpretation or software assisted interpretation of mass spectra are performedsoftware assisted interpretation of mass spectrasoftware assisted interpretation of mass spectra A recent technique for structure elucidation in mass spectrometry, called precursor ion fingerprinting identifies individual pieces of structural information by conducting a search of the tandem spectra of the molecule under investigation against a library of the product-ion spectra of structurally characterized precursor A recent technique for structure elucidation in mass spectrometry, called precursor ion fingerprinting identifies individual pieces of structural information by conducting a search of the tandem spectra of the molecule under investigation against a library of the product-ion spectra of structurally characterized precursortandem spectra product-ion spectratandem spectra product-ion spectra

Mass spectrometry

Techniques Techniques Of mass spectrometry GC –MS : GC –MS : 1.Definitive method to quantify standard reference materials 2.Identifying trace contaminants or toxins 3.Identification of drugs

Techniques Techniques Of mass spectrometry HPLC –MS : HPLC –MS : 1.Screening & confirmation in inborn errors of metabolism 2.Analysis of amino acids

MALDI

SELDI-TOF

Tandem Mass Spectrometry A tandem mass spectrometer is a mass spectrometer that has more than one analyser, usually two, with a collision chamber in between.

MS/MS uses 2 stages of mass analysis: 1. Selection of an ion 2. Analysis of ion fragments produced by collision with inert gas.

Phe Subs B

Proteomics and mass spectrometry APPLICATIONS OF mass spectrometry Pharmacokinetics New born screening

applications of mass spectrometry applications of mass spectrometry to study proteins Best two techniques: Best two techniques: ESI ESI MALDI MALDI Using two approaches: Top-Down Top-Down Bottom-Up Bottom-Up

Newborn screening (NBS)

MS/MS allows the screening of a numerous array of metabolic disorders in a single analytical run: Amino Acid Disorders PKU, MSUD, Tyr, Cit, etc Fatty Acid Oxidation Disorders MCAD, VLCAD, LCHAD, etc. Organic Acid Disorders PA, MMA, IVA, GA-1, etc

% intensity

Advantages of MS/MS as a screening tool in NBS  Sensitive  Specific  Accurate Quantitation  Internal standards: gold standard for accuracy  High impact  Multiple Metabolite, Multiple Disease Screening  cost effective  High throughput

applications of mass spectrometry applications of mass spectrometry to study proteins Best techniques: Best techniques: ESI - Quadrupole ESI - Quadrupole MALDI-TOF MALDI-TOF SELDI-TOF SELDI-TOF

TOP-DOWN approach intact proteins are ionized by either of the three techniques described above, and then introduced to a mass analyzer

BOTTOM-UP approach Bottom-up proteomics is a common method to identify proteins and characterize their amino acid sequences and post-translational modifications by proteolytic digestion of proteins prior to analysis by mass spectrometry. proteomicspost-translational modificationsproteolytic digestionmass spectrometryproteomicspost-translational modificationsproteolytic digestionmass spectrometry

applications of mass spectrometry applications of mass spectrometry to study proteins  Identifying unknown proteins  Protein sequencing ( peptides are sequenced by generating multiple sets of peptides)

applications of mass spectrometry applications of mass spectrometry to study proteins  Identification of chemical modification ( post-translational modification in proteins after synthesis)  Identification of organisms (identifying bacteria by finger printing proteins)

Automated sample preparation Automated sample injection High throughput MS/MS One 3/16thin. blood disk Butyl ester derivatization for enhance sensitivity Batched microtiter plate Automated sample handling and preparation Two minute analysis time Microtiter plate format Small sample volume 10 μL Automated injection Low sample carry-over Enhanced sensitivity Robust, sensitive, low maintenance systems 120,000 samples /year Automated MS generation Automated interpretation & reporting