Protein identification
(Peptide mass fingerprinting)
Dr. Aamir Shehzad
Senior Scientist,
Drug Discovery and Structural Biology Lab,
Health Biotechnology Division (HBD),
NIBGE
Proteomics is the study of the proteome, the protein complement of the genome.
The proteome refers to all proteins that are expressed by a cell, tissue or organism under
defined conditions, at a certain time.
Not all the genes are expressed in all the cells.
Proteins essential to basic cellular functions -
expressed in virtually all cells.
Proteins with highly specialized functions - expressed
only in specific cell types.
Rhodopsin
Longer amino acid sequences often form domains, which confer specific properties or
functions on a protein.
The occurrence of motifs and domains in proteins of unknown function offer hints
as to their cellular roles.
Analytical
Protein Sequence Function
proteomics
Protein fractionation:
Sample preparation typically involves multidimensional protein fractionation to reduce
sample complexity
There are three principal separation approaches used with intact proteins are 1D- and 2D-
SDS-PAGE and isoelectric focusing (IEF).
Other alternatives are chromatographic techniques (reverse phase (RP), size exclusion,
ion exchange, or affinity chromatography).
One-Dimensional SDS-PAGE:
In 1D-SDS PAGE, the protein sample is dissolved in a loading buffer that usually contains a
thiol reductant (mercaptoethanol or DTT) and SDS.
The separation method is based on the binding of SDS to the protein, which imparts
negative charge (from the SDS sulfate group) to the protein.
When the gel is subjected to high voltage, the protein-SDS complexes migrate through the
cross-linked polyacrylamide gel at rates based on their molecular weights.
The bands that appear to contain a single protein may actually contain multiple molecular
species.
Two-dimensional SDS-PAGE:
Two-dimensional SDS-PAGE is actually a combination of two different types of separations. In
the first, the proteins are resolved on the basis of isoelectric point by lEF.
In the second, focused proteins then are further resolved by electrophoresis on a
polyacrylamide gel based on their molecular weights.
Immobilized pH gradient (IPG) strips are used for IEF. The IPG strip is based on the use of
immobilized pH gradients, in which polycarboxylic acid ampholytes are immobilized on
supports to reproducibly create stable pH gradients.
The strip is hydrated with a buffer and the protein is slowly loaded into the strip under
voltage.
After the focusing step, the strip is treated with a buffer that contains a thiol reductant and
SDS and then is joined to the SDS-PAGE slab gel.
Why Digest Proteins?
MS instruments cannot accurately measure the mass of intact proteins. The greater the
mass of the protein, the greater the error.
Better assignment of masses to peptides.
Ideally, the length of peptide fragments should be 6-20 amino acids for MS analysis and
database matching.
Peptides shorter than about 6 amino acids generally are too short to produce unique
sequence matches in database searches.
Trypsin
Trypsin cleaves proteins at lysine and arginine residues, unless either of these is followed
by a proline.
High activity, selectivity and relatively low cost.
Trypsin will cut proteins more frequently than will a protease that cuts at only one amino
acid residue. As a general rule, a 50 kDa protein will yield about 30 tryptic peptides.
For example, 56 % of the tryptic peptides in yeast are less than 6 amino acids long.
For more comprehensive proteome sequence coverage, alternative proteases are often
used in combination.
Glu-C:
Cleaves at the carboxyl side of glutamate and aspartate.
Different cleavage specificity than trypsin.
Useful for analysis of proteins with regions of high lysine and arginine content.
Chymotrypsin:
Cleaves at aromatic amino acids (tyrosine, phenylalanine, and tryptophan).
Chemicals such as cyanogen bromide (CNBr):
Cleaves proteins at methionine
Infrequency of methionine residues in most proteins, thus yields relatively few, large
fragments.
Mass spectrometry (MS) techniques
Matrix-assisted laser desorption/ionization time-of-flight – MS
Electrospray Ionization- MS
Inductively coupled plasma – MS
Quadrupole ion trap - MS
Peptide Mass Fingerprint
Myoglobin
GLSDGEWQQV LNVWGKVEAD IAGHGQEVLI
RLFTGHPETL EKFDKFKHLK TEAEMKASED
LKKHGTVVLT ALGGILKKKG HHEAELKPLA
QSHATKHKIP IKYLEFISDA IIHVLHSKHP
GDFGADAQGA MTKALELFRN DIAAKYKELG
FQG
Peptide Mass Fingerprint
• Myoglobin digested with Trypsin prtoease
GLSDGEWQQV LNVWGKVEAD IAGHGQEVLI
RLFTGHPETL EKFDKFKHLK TEAEMKASED
LKKHGTVVLT ALGGILKKKG HHEAELKPLA
QSHATKHKIP IKYLEFISDA IIHVLHSKHP
GDFGADAQGA MTKALELFRN DIAAKYKELG
FQG
Trypsin: digestion enzyme
Highly specific
Cuts after Lysine & Arginine except if followed by Proline
Peptide Masses
1811.90 GLSDGEWQQVLNVWGK
1606.85 VEADIAGHGQEVLIR
1271.66 LFTGHPETLEK
1378.83 HGTVVLTALGGILK
1982.05 KGHHEAELKPLAQSHATK
1853.95 GHHEAELKPLAQSHATK
1884.01 YLEFISDAIIHVLHSK
1502.66 HPGDFGADAQGAMTK
748.43 ALELFR
ALELFR
LFTGHPETLEK
HGTVVLTALGGILK
HPGDFGADAQGAMTK
VEADIAGHGQEVLIR
GLSDGEWQQVLNVWGK
Peptide Mass Fingerprint
GHHEAELKPLAQSHATK
YLEFISDAIIHVLHSK
KGHHEAELKPLAQSHATK
Peptide Mass Fingerprint Analysis
• Protein sequence from sequence database
– In silico digest
– Mass computation
• For each protein sequence in turn:
– Compare computer generated masses with observed
spectrum
Sample Preparation for MS/MS
Enzymatic Digest
Single Stage MS
MS
Tandem Mass Spectrometry
(MS/MS)
Precursor selection
Tandem Mass Spectrometry
(MS/MS)
Precursor selection +
collision induced dissociation
(CID)
MS/MS
Tandem Mass Spectrometry
(MS/MS)
Peptide Fragmentation for Protein Sequencing
Peptides consist of amino-acids
arranged in a linear backbone.
yn-i
yn-i-1
-HN-CH-CO-NH-CH-CO-NH-
Ri CH-R’
i+1
bi R”
i+1
bi+1
collision induced dissociation
Example: Fragmentation of a peptide (4 amino acids)
Example: Fragmentation of a peptide (4 amino acids)
Example: Fragmentation of a peptide (4 amino acids)
Peptide Fragmentation
Peptide: S-G-F-L-E-E-D-E-L-K
MW ion ion MW
88 b1 S GFLEEDELK y9 1079
145 b2 SG FLEEDELK y8 1022
292 b3 SGF LEEDELK y7 875
405 b4 SGFL EEDELK y6 762
534 b5 SGFLE EDELK y5 633
663 b6 SGFLEE DELK y4 504
778 b7 SGFLEED ELK y3 389
907 b8 SGFLEEDE LK y2 260
1020 b9 SGFLEEDEL K y1 147
Peptide Fragmentation
100
% Intensity
0 m/z
250 500 750 1000
Peptide Fragmentation
88 145 292 405 534 663 778 907 1020 1166 b ions
S G F L E E D E L K
1166 1079 1022 875 762 633 504 389 260 147 y ions
y6
100
y7
% Intensity
y5
b3
b4
y2 y3 y4 b 5 b8 y
b6 b7 b9 8 y9
0 m/z
250 500 750 1000
Peptide Identification
Peptide Sequence Identification
Given:
• The mass of the precursor ion, and
• The MS/MS spectrum
Output:
• The amino-acid sequence of the peptide
Protein Identification – Database
searching
MASCOT: an MS data based search engine
MASCOT: an MS data based search engine
Protein Identification – Database
searching
Protein Identification – Database
searching
Protein Identification – Database
searching
Protein Identification – Database
searching
MASCOT: an MS data based search engine
