MS Principles

Dwight Glenn Jr. Briana Halbert

What is Mass Spectroscopy?

Mass Spectrometry (MS) is a technique for

measuring the mass, (MW) of a molecule. From this information it is possible to gain structural information about a molecule by measuring the mass fragments produced when molecules are broken apart Mass Spectrometers have 3 basic parts: Ionization source, Mass analyzer, and Detector

Mass Spectrometry Needs

Ionization-how the protein is injected in to the

MS machine Separation-Mass and Charge is determined Activation-protein are broken into smaller fragments (peptides/AAs) Mass Determination-m/z ratios are determined for the ionized protein fragments/peptides

Mass spectrometers
ion source

Linear Time Of Flight tube

Time of flight (TOF) (MALDI) Measures the time required for ions to fly down the length of a chamber. Often combined with MALDI (MALDI-TOF) Detections from Reflector Time Of Flight tube multiple laser bursts are averaged. Multiple laser
ion source

detector

time of flight

Tandem MS- MS/MS -separation and identification of compounds in complex mixtures - induce fragmentation and mass analyze the fragment ions. - Uses two or more mass analyzers/filters separated by a collision cell filled with Argon or Xenon
detector

reflector

time of flight

Different MS-MS configurations

Quadrupole-quadrupole (low energy) Magnetic sector-quadrupole (high) Quadrupole-time-of-flight (low energy) Time-of-flight-time-of-flight (low energy)

How does a mass spectrometer work?

Create ions

Separate ions
Mass analyzer
MALDI-TOF

Detect ions

Ionization

method
MALDI Electrospray
(Proteins must be charged and dry)

MW

Triple Quadrapole

AA seq

MALDI-QqTOF

Mass spectrum Database analysis

AA seq and MW

QqTOF

AA seq and protein modif.

Three Components:

This is a glimpse to how a mass spectrometer works

starting from the sample to the data on the display screen.

Mass Spec Principles

Sample

+ _

Ionizer

Mass Analyzer

Detector

MS

Video
http://www.youtube.com/watch?v=J-wao0O0_qM

Typical Mass Spectrometer

What happens inside the spectrometer?

During an experiment a small amount of a sample

is vaporized into the ionization source. The energy of the electron beam will strike the organic molecule and will dislodge a valence electron This dislodgement produces a cation radical

Cation Radical
Cation because the molecule has lost an

electron and has a positive charge Radical because the molecule now has an odd number of electrons

Next Step
Electron bombardment transfers so much energy

that most of the cation radicals fragment after they are formed. This means that they break into smaller pieces because a bond has been weakened by loss of an electron and some retain their positive charge while some are neutral. These fragments then flow through a curved pipe in a strong magnetic field

What is the data?

In the pipe there are different paths and

according to the mass-to-charge ratio (m/z) it will determine which fragments go where. Lower mass charge ratio is deflected more. Neutral fragments (radical) are not deflected by the magnetic field and are then lost on the walls of the pipe. Positively charged fragments (cation) are sorted by the mass spectrometer onto a detector which records them as peaks at various (m/z) ratios

How to read the data:

Since the number of charges z on each ion is

usually 1, the value of m/z for each ion is its mass. The mass spectrum of a compound is typically presented as a bar graph (m/z) is on the x-axis Relative abundance is on the y axis.

How to read the data

Some important peaks to remember: The base peak is the peak assigned to the intensity of 100% It is also the tallest peak, is the most stable cation

How to read the data

The next peak is the parent peak This peak corresponds to the unfragmented cation radical This is also known as the molecular ion

What is it?
Since the parent peak has a m/z of 44 that means

that the MW of the substance will equal 44. The mass spectrum shown was for propane C3H8 has a molecular weight of 44

Video
http://www.youtube.com/watch?v=tOGM2gOHKP

c&NR=1&feature=endscreen

Question:
What is mass spec used for.

Answer:
Measuring the mass, (MW) of a

molecule

Question
The base peak in a mass spectrum represents

(a) the most stable radical (b) the most stable alkene (c) the most stable cation (d) the molecular ion

Answer
C. the most stable cation