Mass Spectrometry Flashcards
Mass Spectrometry is a powerful and sensitive analytical technique that is used to:
Determine the ___ of atoms, isotopes, molecules, or molecular fragments
Determine the ___ of molecules and complex mixtures
IDENTIFY unknown compounds
QUANTIFY known materials
detection at 10^-12 g or 10^-15 moles
identification at low concentrations ~1 ppt
masses
elemental composition
an instrument that SEPARATES RAPIDLY MOVING IONS based upon their ___;
allows the masses of individual atoms or molecules
that have been converted to ions to be determined.
The units used for Mass spectrometry is ___
Mass spectrometers can be MICROWAVE to ROOM szie
MASS-to-CHARGE (m/z) ratio
Daltons (Da)
Traits of mass spectrometer (4)
produces ions,
separates them according to their m/z values,
detects the ions and
plots the mass spectrum.
A mass spectrum is a graph of ___ or relative abundance as a function of mass to charge ratio (m/z)
Mass spectra are usually depicted either as a SIMPLE HISTOGRAM or as the exact graph of intensity versus m/z ratio
Under appropriate conditions, a mass spectrum can
provide the ____ and the ____ of the compound being analyzed.
ion intensity
MOLECULAR MASS
STRUCTURE
MS Sources: (PLS MEMORISE
ATOMS
ICP
ATOMS
ICP - Inductively-Coupled Plasma
MS Sources: (PLS MEMORISE
SMALL MOLECULES
EI
CI
FI
APCI
APPI
MS Sources: (PLS MEMORISE
SMALL MOLECULES
EI - Electron Ionization
CI - Chemical Ionization
FI - Field Ionization
APCI - Atmospheric Pressure Chemical Ionization
APPI - Atmospheric Pressure Photoionization
MS Sources: (PLS MEMORISE
LARGE MOLECULES
FAB
MALDI
ESU
MS Sources: (PLS MEMORISE
LARGE MOLECULES
FAB - Fast Atom Bombardment
MALDI - Matrix-Assisted Laser Desorption/Ionization
ESU - Electrospray Ionization
Electrospray Ionization parts
Electrospray
Laser (Focusing Lens)
Sample
Mass Analyzer
MALDI - Matrix-Assisted Laser Desorption/Ionization Parts (2)
337 nm UV laser
Sample/matrix crystals
Pharmaceutical analysis in Mass Spectrometry (7)
Pharmaceutical analysis
Bioavailability studies
Drug metabolism studies, pharmacokinetics
Characterization of potential drugs
Drug degradation product analysis
Screening of drug candidates
Identifying drug targets
Biomolecule characterization in Mass Spectrometry (2)
Proteins and peptides
Oligonucleotides
Environmental analysis in Mass Spectrometry (2)
Pesticides on foods
Soil and groundwater contamination
MATRIX-ASSISTED LASER DESORPTION / IONIZATION (MALDI) applied to ___ is a powerful technology for analyzing spatial arrangement of molecules in THIN TISSUE SECTIONS
MASS SPECTROMETRY IMAGING (MSI)
TISSUE IMAGING MASS SPECTROMETRY can be used to
determine the distribution of hundreds or even thousands of UNKNOWN ANALYTES in a single experiment
such as:
proteins,
peptides,
lipids,
drugs, or metabolites.
Applications for mass spectrometry imaging include: (4)
profiling of ENDOGENOUS BIOMELECULES within tissues/organs,
comparisons of ANALYTE ABUNDANCES between samples taken at DIFFERENT TIMES or after different treatments,
analysis of the distribution and METABOLISM of dosed drugs.
General Approaches using MS (5)
Step 1: ISOLATE CELL or other protein source
Step 2: LYSE CELLS and ISOLATE PROTEINS
Step 3: BREAK UP PROTEINS into smaller (but still
relatively large) amino acid chains
Step 4: SEPARATE CHAINS (2D gel, gas or liquid
chromatography)
Step 5: ANALYZE SEPARATED PROTEIN PARTS by mass spectrometry
Classical analytical methods for protein Characterization:
Simple, inexpensive, fast
Crude measure of MOLECULAR WEIGHT and PURITY
w/Blotting - Sensitive & Selective detection
Polyacrylamide Gel Electrophoresis (PAGE)
Classical analytical methods for protein Characterization:
Used for MAPPING DISEASE MARKERS
Variety of pH gradients
Automated, high throughput instruments
Isoelectric Focusing (IEF)
Classical analytical methods for protein Characterization:
Orthogonal separations - alrge separation space
Detection of SMALL changes in COMPLEX samples
Separation of POST-TRANSLATIONALLY MODIFIED proteins
Dynamic Range PROBLEMS DUE TO SAMPLE LOADING CAPACITY
Two Dimensional IEF - PAge (2D Gels
Isoelectric focusing:
This separates proteins based on ISOELECTRIC POINT
The isoelectric point is the pH at which the protein has NO NET CHARGE
pH gradients may be large 2-10 or small 6-7
Typically this is done with an immobilized pH
___or with a TUBE GEL containing a low concentration of ___.
___ are added to create a pH gradient in an electric field and the proteins are loaded.
The IEF gel is placed in an electrophoresis system for up to ___ and the proteins form tight bands at their isoelectric point.
The IEFgels are now ready for the second method.
GRADIENT GEL STRIP
POLYACRYLAMIDE
AMPHOLYTES
24 hours
2D-GE - Polyacrylamide Gel Electrophoresis:
Separation of proteins on basis of SIZE
SMALL PROTEINS migrate through gel matrix QUICKEST
Resulting gel has proteins separated
Horizontally by ___
Vertically by ___
The IEF gel is soaked in a solution containing chemical to denature the proteins which gives the proteins a net negative charge. This means that all proteins will move in one direction.
The IEF gel is then put in the one long well in the stacking gel, sealed in place with AGAROSE, and the proteins subjected to an electric field to separate.
The larger proteins are found at the top and the smaller ones are found at the bottom of the gel. proteins appear as SPOTS which can be used for mass spectrometry directly
Horizontally by IEP
Vertically by size
Further processing of Polyacrylamide Gel Electrophoresis includes ___, ___, and ___
spot excision
Trypsin digiestion
Mass spectrometry
Liquid Chromatography:
Proteins washed through ___ (or
columns)
Separates based on specific properties (3)
Depends on column matrix/eluent
Usually 2 (or more) columns used
Can be coupled to Mass Spec (online)
Or fractions collected for later analysis (offline)
Example:
capillary column
Charge
Size
Hydrophobicity
MudPIT (Multidimensional Protein
Identification Technology)
____ is an isobaric labeling method used in
quantitative proteomics by tandem
mass spectrometry to determine the
AMOUNT OF PROTEINS FROM DIFFERENT SOURCES in a single experiment.
Isobaric tags for relative and
absolute quantitation (iTRAQ)
The end of protein MS studies
From PROTEOME to dB protein genome est and then Gene product I.D.
Protein Identification
___ cut at defined sites and this will provide a series of ___ with different masses, these are unique to the protein
Peptide Mass Fingerprinting
Proteases
Peptides
Protein Identification
From the fact that Peptides fragment in a predictable way
A peptide of ____ should be sufficient to
uniquely identify a protein
MS/MS
> 7 amino acids
