Mass Spectrometry

Question 1

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

Answer 1

masses

elemental composition

Question 2

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

Answer 2

MASS-to-CHARGE (m/z) ratio

Daltons (Da)

Question 3

Traits of mass spectrometer (4)

Answer 3

 produces ions,
 separates them according to their m/z values,
 detects the ions and
 plots the mass spectrum.

Question 4

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.

Answer 4

ion intensity

MOLECULAR MASS

STRUCTURE

Question 5

MS Sources: (PLS MEMORISE

ATOMS
ICP

Answer 5

ATOMS
ICP - Inductively-Coupled Plasma

Question 6

MS Sources: (PLS MEMORISE

SMALL MOLECULES
EI
CI
FI
APCI
APPI

Answer 6

MS Sources: (PLS MEMORISE

SMALL MOLECULES
EI - Electron Ionization
CI - Chemical Ionization
FI - Field Ionization
APCI - Atmospheric Pressure Chemical Ionization
APPI - Atmospheric Pressure Photoionization

Question 7

MS Sources: (PLS MEMORISE

LARGE MOLECULES
FAB
MALDI
ESU

Answer 7

MS Sources: (PLS MEMORISE

LARGE MOLECULES
FAB - Fast Atom Bombardment
MALDI - Matrix-Assisted Laser Desorption/Ionization
ESU - Electrospray Ionization

Question 8

Electrospray Ionization parts

Answer 8

Electrospray
Laser (Focusing Lens)
Sample
Mass Analyzer

Question 9

MALDI - Matrix-Assisted Laser Desorption/Ionization Parts (2)

Answer 9

337 nm UV laser

Sample/matrix crystals

Question 10

Pharmaceutical analysis in Mass Spectrometry (7)

Answer 10

Pharmaceutical analysis
 Bioavailability studies
 Drug metabolism studies, pharmacokinetics
 Characterization of potential drugs
 Drug degradation product analysis
 Screening of drug candidates
 Identifying drug targets

Question 11

Biomolecule characterization in Mass Spectrometry (2)

Answer 11

 Proteins and peptides
 Oligonucleotides

Question 12

Environmental analysis in Mass Spectrometry (2)

Answer 12

 Pesticides on foods
 Soil and groundwater contamination

Question 13

MATRIX-ASSISTED LASER DESORPTION / IONIZATION (MALDI) applied to ___ is a powerful technology for analyzing spatial arrangement of molecules in THIN TISSUE SECTIONS

Answer 13

MASS SPECTROMETRY IMAGING (MSI)

Question 14

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:

Answer 14

 proteins,
 peptides,
 lipids,
 drugs, or metabolites.

Question 15

Applications for mass spectrometry imaging include: (4)

Answer 15

 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.

Question 16

General Approaches using MS (5)

Answer 16

 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

Question 17

Classical analytical methods for protein Characterization:

Simple, inexpensive, fast
Crude measure of MOLECULAR WEIGHT and PURITY
w/Blotting - Sensitive & Selective detection

Answer 17

Polyacrylamide Gel Electrophoresis (PAGE)

Question 18

Classical analytical methods for protein Characterization:

Used for MAPPING DISEASE MARKERS
Variety of pH gradients
Automated, high throughput instruments

Answer 18

Isoelectric Focusing (IEF)

Question 19

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

Answer 19

Two Dimensional IEF - PAge (2D Gels

Question 20

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.

Answer 20

GRADIENT GEL STRIP

POLYACRYLAMIDE

AMPHOLYTES

24 hours

Question 21

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

Answer 21

 Horizontally by IEP
 Vertically by size

Question 22

Further processing of Polyacrylamide Gel Electrophoresis includes ___, ___, and ___

Answer 22

spot excision

Trypsin digiestion

Mass spectrometry

Question 23

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:

Answer 23

capillary column

 Charge
 Size
 Hydrophobicity

MudPIT (Multidimensional Protein
Identification Technology)

Question 24

____ 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.

Answer 24

Isobaric tags for relative and
absolute quantitation (iTRAQ)

Question 25

The end of protein MS studies

Answer 25

From PROTEOME to dB protein genome est and then Gene product I.D.

Question 26

Protein Identification

___ cut at defined sites and this will provide a series of ___ with different masses, these are unique to the protein

Answer 26

Peptide Mass Fingerprinting

Proteases
Peptides

Question 27

Protein Identification

From the fact that Peptides fragment in a predictable way

A peptide of ____ should be sufficient to
uniquely identify a protein

Answer 27

MS/MS

> 7 amino acids