Unknown

protein in
sample

MALDI-TOF for Protein

Identification

Trypsinated
peptides

Experimental
MS spectrum

+
+

m/z

Trypsinated peptides
+ matrix
Sequence
analysis using
Mascot search
engine

Unknown protein could

possibly a protein B

Protein Database (MS library)

Gene sequence
Protein sequence (theoretical)

Protein A
AYMKMADERAPELEKEEVGVAA

Protein B
VAVREEVAKQEGMQKSRTALQV

Protein C
AEEKKAPRRNVGGARDPVKVVT

Peptide Mass
Fingerprinting (PMF)

Artificially
trypsinated
peptides
AYMK
MADER
APELEK
EEVGVAA

Artificial
MS spectra
built
25%
matched
m/z

VAVR
EEVAK
QEGMQK
SRTALQV

75%
matched
m/z

AEEK
KAPRR
NVGGAR
DPVKVVT

0%
matched
m/z

ESI-MS/MS for Protein Sequencing

Unknown
protein in
sample

Trypsinated
peptides

Ionized
peptides
+
+
+
+
+

Experimental
MS spectrum

Full-scan mode
Only one analyzer involved in full scan
mode to separate peptides base on m/z

Switch to MS-MS mode for analysis of selected ion (peptide)

MS-MS mode

Mass analyzers
1 Select ion of interest (parent ion)
2 Allow collision to generate ion fragments (daughter ions)
3 Separate ion fragments base on m/z
+

Collision

+
+

Dissociate bonding
along peptides

Experimental
MS/MS spectrum
y2
b2

b3
y1

Selected parent
ion (peptides with
4 amino acids)

b1

m/z

+
+

m/z

+
+

+
+

y3
b1
y2
b2
y1
b3
Potential ion fragments with
differential m/z

m/z

+
+

m/z

y3

ESI-MS/MS for Protein Sequencing

Protein Database (MS/MS library)
Gene sequence

Protein sequence (theoretical)

Protein A
AYMKMADERAPELEKEEVGVAA
Artificially trypsinated peptides
MADER

AYMK

APELEK

EEVGVAA

A PELEK
AP ELEK
APE LEK
APEL EK
APELE K

E EVGVAA
EE VGVAA
EEV GVAA
EEVG VAA
EEVGV AA
EEVGVA A

Hypothetic peptide fragments

b1
b2
b3

A YMK
AY MK
AYM K

M ADER
MA DER
MAD ER
MADE R

y3
y2
y1

Theoretically
derived mass
spectrum

Artificial MS/MS spectra built

b1
b2

y3
y2
y1
b3

m/z

m/z

m/z

m/z

m/z

m/z

m/z

m/z

Protein B

m/z

m/z

Protein C

m/z

m/z

ESI-MS/MS for Protein Sequencing

Unknown protein
in sample

Experimental MS/MS spectrum

Sequence
analysis using
Sequest
search engine

Unknown protein could

possibly a protein B with
known matched sequence

VAVREEVAKQEGMQKXXXXXXX
Protein Database (MS/MS library)
25% matched
Protein A

AYMK

MADER

Protein B

75% matched

VAVR

EEVAK

Protein C

AEEK

APELEK

EEVGVAA

QEGMQK

SRTALQV

NVGGAR

DPVKVVT

0% matched

KAPRR

Questions to check for

understanding
6. Mass spectrometry (MS) is an
analytical method widely used for
chemical compound analysis lately.
Briefly describe the important
components of a mass spectrometer
for protein sequencing.
7. What do you understand the
method tandem mass
spectrometry?