What is mass spectrometry?
• Analytical method which measures the mass/charge
ratio and intensity of ions (and fragments) in the gas
phase
• Precise (<1 part per million mass accuracy)
• Sensitive (<10-15 mol)
• Fast (µ sec)
• Unlimited mass range
• >6 orders of magnitude dynamic range
• No special reagents needed (low cost/sample)
• Generic
• Sample must be salt-free and relatively pure
• Hyphenation usually needed eg. GC-MS, LC-MS

All mass spectrometers consist of 3 basic
components:
ION SOURCE
- gas phase ions are
produced
MASS FILTER
- ions of different
m/z are separated
either in space or in
time
DETECTOR
- ions generate an
electrical signal

All mass spectrometers consist of 3 basic
components:
ION SOURCE
- gas phase ions are
produced
MASS FILTER
- ions of different
m/z are separated
either in space or in
time
DETECTOR
- ions generate an
electrical signal
• Positively charged droplets
form at the spray tip
• Droplets are drawn into a
heated capillary in a stream of
nitrogen
• Desolvation occurs via a series
of Coulomb explosions
• Gas phase ions are produced
Electrospray

All mass spectrometers consist of 3 basic
components:
ION SOURCE
- gas phase ions are
produced
MASS FILTER
- ions of different
m/z are separated
either in space or in
time
DETECTOR
- ions generate an
electrical signal
Time of Flight

All mass spectrometers consist of 3 basic
components:
ION SOURCE
- gas phase ions are
produced
MASS FILTER
- ions of different
m/z are separated
either in space or in
time
DETECTOR
- ions generate an
electrical signal
Micro Channel Plate

MS workflow
Sample prep (slow) data acquisition (fast) data analysis (slow)

Tryptic digest MSMS workflow
Sample prep Data acquisition Data analysis
Data dependent
peptide
fragmentation.
Less than 1 second
per peptide fragment
spectrum. Data
acquired over 16 min
HPLC run.
(automated)
Compound extraction,
charge deconvolution
and mgf. file generation
(automated, 30 sec)
2. SDS-PAGE (60 min)
4. Trypsin digestion
(16 hours)
5. In-line reversed
phase HPLC
(automated, 16 min)
1. Protein
purification (days)
Submission to batch
queue and 3-phase
Mascot search
(automated, 3 min)
Manual data evaluation,
annotation and input
into Scarab (2-3 min/
sample)
E-mail notification
3. Reduction-alkylation
(90 min)

Protein intact mass workflow
Sample prep Data acquisition Data analysis
Data acquired over 2
min HPLC run.
(automated)
Compound extraction,
charge deconvolution
(automated, 30 sec)
2. In-line reversed
phase desalting
(automated, 2 min)
1. Protein
purification (days)
Manual data evaluation
Comparison between
observed and expected
mass from sequence
Identification of mass
discrepancies
PTMs (Unimod)
Truncations (PAWS)

Protein native MS workflow
Sample prep Data acquisition Data analysis
Manual direct
infusion via syringe
pump
(20 min)
Manual data analysis
1. Monomer mass
2. Non-covalent
interactions
3. Charge assignment
4. Multimeric state
5. Charge distribution
analysis
(conformational
states)
6. Charge radius
(hours)
2. Off-line desalting
via size exclusion
spin column x 3
(30 min)
1. Protein
purification (days)