PROTEIN SEQUENCING

In 1953, Frederick Sanger of Cambridge University in England reported the amino

acid sequences of the two polypeptide chains composing the protein insulin. Not only was
this a remarkable achievement in analytical chemistry but it helped to demystify speculation
about the chemical nature of proteins. Sangers results clearly established that all of the
molecules of a given protein have a fixed amino acid composition, a defined amino acid
sequence, and therefore an invariant molecular weight. In short, proteins are well defined
chemically. Today, the amino acid sequences of some 100,000 different proteins are known.
Although many sequences have been determined from application of the principles first
established by Sanger, most are now deduced from knowledge of the nucleotide sequence of
the gene that encodes the protein.
Protein Sequencing Strategy
The usual strategy for determining the amino acid sequence of a protein involves
eight basic steps:
1. If the protein contains more than one polypeptide chain, the chains are separated and
purified.
2. Intrachain S--S (disulfide) cross-bridges between cysteine residues in the polypeptide chain
are cleaved. If these disulfides are interchain linkages, then step 2 precedes step1.
3. The amino acid composition of each polypeptide chain is determined.
4. The N-terminal and C-terminal residues are identified.
5. Each polypeptide chain is cleaved into smaller fragments.
6. Sequence determination of peptide fragments.
7. The overall amino acid sequence of the protein is reconstructed from the sequences in
overlapping fragments.
8. The positions of S--S cross-bridges formed between cysteine residues are located.
Each of these steps is discussed in greater detail in the following sections.
Step 1: Separation of Polypeptide Chains:
If the protein of interest is a heteromultimer (composed of more than one type of
polypeptide chain), then the protein must be dissociated and its component polypeptide
subunits must be separated from one another and sequenced individually. Subunit
associations in multimeric proteins are typically maintained solely by noncovalent forces, and
therefore most multimeric proteins can usually be dissociated by exposure to pH extremes, 8
M urea, 6 M guanidinium hydrochloride, or high salt concentrations. (All of these treatments
disrupt polar interactions such as hydrogen bonds both within the protein molecule and
between the protein and the aqueous solvent.) Once dissociated, the individual polypeptides
can be isolated from one another on the basis of differences in size and/or charge.

Occasionally, heteromultimers are linked together by interchain S--S bridges. In such

instances, these cross-links must be cleaved prior to dissociation and isolation of the
individual chains. The methods described under step 2 are applicable for this purpose.
Step 2: Cleavage of Disulfide Bridges
A number of methods exist for cleaving disulfides. An important consideration is to
carry out these cleavages so that the original or even new S--S links do not form. Oxidation
of a disulfide by performic acid results in the formation of two equivalents of cysteic acid.
Because these cysteic acid side chains are ionized SO3 groups, electrostatic repulsion (as well
as altered chemistry) prevents S--S recombination. Alternatively, sulfhydryl compounds such
as 2-mercaptoethanol readily reduce S--S bridges to regenerate two cysteine-SH side chains.
However, these SH groups recombine to re-form either the original disulfide link or, if other
free Cys-SHs are available, new disulfide links. To prevent this, SOS reduction must be
followed by treatment with alkylating agents such as iodoacetate or 3-bromopropylamine,
which modify the SH groups and block disulfide bridge formation.

Step 3: Analysis of Amino Acid Composition

Amino acid composition of polypeptide chain is determined using amino acid

analyzer. The amino acid analyzer consists of ion-exchange column through which the
appropriate elution buffer is pumped after the amino acids are introduced at the top of the
column with the help of digested sample and buffer. As the separated amino acid emerge,
they are mixed with ninhydrin solution and passed through heated coil of tubing to allow the
formation of the colored ninhydrin product. The separated ninhydrin color products then pass
through a cell that measures the optical absorbance at 540 and 440 nm and plots the results on
a strip-chart recorder. The absorbance is measured at two wavelengths because proline,
which is substituted at its amino group, forms a different ninhydrin color product with an
absorption maximum that is correspondingly different from that pf the remaining amino
acids.
Usually the amino acid analyzer is first standardized by running through it a sample
containing known quantities of amino acids to account for any differences in their ninhydrin
reaction properties. In this way it is possible to relate directly the amount amino acid present
to the amount of colored product formed, as measured by the area under peak produced on
the strip chart recorder. Similarly, the amino acid hydrolysate of a protein of unknown
composition can be run through the analyzer and relative peak areas can be used to estimate
the ratios of different amino acids present.

Step 4: Identification of the N- and C-Terminal Residues

End-group analysis reveals several things. First, it identifies the N- and C-terminal
residues in the polypeptide chain. Second, it can be a clue to the number of ends in the
protein. That is, if the protein consists of two or more different polypeptide chains, then more
than one end group may be discovered, alerting the investigator to the presence of multiple
polypeptides.

N-Terminal analysis:
Amino terminal end of polypeptide chain determined by either of three methods
namely sangar method, Edmanns method and dansyl chloride method. In sanger method,
Fluorodinitrobenzene used. Polypeptide reacts with FDNB to form Dinitrophenol complex
with polypeptide. Subsequent analysis releases a colored dinitrophenol labeled amino
terminal amino acid, which can be identified by its characteristic migration rate on thin-layer
chromatography or paper electrophoresis. In Edman degradation method, Phenyl isothio
cyanate (PIT) is used as reagent. First the polypeptide is rected with phenyl isothio cyanate
to forma polypeptidyl phenylthiocarbamyl derivative. Gentle hydrolysis releases the amino
terminal amino acid as a phenylthiohydantoin (PTH), which can be separated and detected
spectrophotometrically. Upto this stage, this method is used to determine N-terminal
aminoacid. This method can also be extended to determine polypeptide sequence. For this,
after the release of PTH, the remaining intact polypeptide, shortened by one amino acid, is
then ready for further cycles of this procedure. Dansyl chloride is also used to determine Nterminal amino acid. This method is more sensitive because of fluorescence measurement.

C- Terminal Analysis:
There are two methods used for C-terminal determination. They are Hydrazine
method and carboxy peptidase method. Chemical methods for carboxy end-group
determination are considerably less satisfactory. Treatment of the peptide with anhydrous
hydrazine at 100*C results in conversion of all the amino acid resiudes to amino acid
hydrazides except for the carboxy-terminal residue, which remains as the free amino acid and
can be isolated and identified chromatographically. Alternatively, polypeptide can be
subjected to limited breakdown with the enzyme carboxypeptidase. Carboxypeptidases are
enzymes that cleave amino acid residues from the C-termini of polypeptides in a successive
fashion. Four carboxypeptidases are in general use: A, B, C, and Y. Carboxypeptidase A
(from bovine pancreas) works well in hydrolyzing the Cterminal peptide bond of all residues
except proline, arginine, and lysine. The analogous enzyme from hog pancreas,
carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues. Thus, a
mixture of carboxypeptidases A and B liberates any C-terminal amino acid except proline.
Carboxypeptidase C from citrus leaves and carboxypeptidase Y from yeast act on any Cterminal residue. Because the nature of the amino acid residue at the end often determines
the rate at which it is cleaved and because these enzymes remove residues successively, care
must be taken in interpreting results. Carboxypeptidase Y cleavage has been adapted to an
automated protocol analogous to that used in Edman sequenators.

Step- 5: Fragmentation of the Polypeptide Chain

The aim at this step is to produce fragments useful for sequence analysis. The cleavage
methods employed are usually enzymatic, but proteins can also be fragmented by specific or
nonspecific chemical means (such as partial acid hydrolysis). Fragmentation can be
achieved by the use of endopeptidases, which are enzymes that catalyze polypeptide chain
cleavage at specific sites in the protein. The specificity of four endopeptidases commonly for
this purpose are Pepsin, Trypsin, Chymotrypsin and Papain. Another specific chemical
method for polypeptide chain cleavage involves reaction with cyanogens bromide. This
reaction cleaves specifically at the methionine residues, with the accompanying conversion of
free carboxyl-terminal methionine to homoserine lactone. For sequencing protein minimum
two proteolytic factors should be selected and two sets of fragements should be prepared.

Step 6: Sequencing Peptide fragments

Peptides resulting from cleavage of the intact protein are generally separated by
column chromatography. The isolated peptides may then be analyzed to determine their
sequence. There two methods recently used for this purpose, they are Edman degradation
and tandem mass spectrometer. Devices called sequenators are available that automate
Edman degradation procedure. The success of these devices depends in large part on the
technical innovation of covalently linking the peptide to be sequenced to glass beads.
Attachment of peptide through its carboxy-terminal group to this immobile phase facilitates
the complete removal of potentially conataminating reaction procedure during successive
stages of the degradation. Thus with the help of this sequenators, peptide fragements are
sequenced.
The other method used for protein sequencing is tandem mass spectrometer. Tandem
MS (or MS/MS) allows sequencing of proteins by hooking two mass spectrometers in
tandem. The first mass spectrometer is used to separate oligopeptides from a protein digest
and then to select in turn each of these oligopeptides for further analysis. A selected ionized
oligopeptide is directed toward the second mass spectrometer; on the way, this oligopeptide is
fragmented by collision with helium or argon gas molecules, and the collection of fragments
is analyzed by the second mass spectrometer. Fragmentation occurs primarily in the peptide
bonds linking successive amino acids in the oligopeptide, so the fragments created represent a
nested set of peptides that differ in size by one amino acid residue. The fragments differ in
mass by 56 atomic mass units (the mass of the peptide backbone atoms (NH-CH-CO)) plus
the mass of the R group at each position, which ranges from 1 atomic mass unit (Gly) to 130
(Trp). MS sequencing has the advantages of very high sensitivity, fast sample processing, and
the ability to work with mixtures of proteins. Subpicomoles (less than 10_12 moles) of
peptide can be analyzed. However, in practice, tandem MS is limited to rather short
sequences (no longer than 15 or so amino acid residues).
Step 7: Reconstruction of the Overall Amino Acid Sequence

The sequences obtained for the sets of fragments derived from two or more cleavage
procedures are now compared, with the objective being to find overlaps that establish
continuity of the overall amino acid sequence of the polypeptide chain. Peptides generated
from specific hydrolysis of the polypeptide can be aligned to reveal the overall amino acid
sequence. Such comparisons are also useful in eliminating errors and validating the accuracy
of the sequences determined for the individual fragments.
Step 8: Location of Disulfide Cross-Bridges
Strictly speaking, the disulfide bonds formed between cysteine residues in a protein are not a
part of its primary structure. Nevertheless, information about their location can be obtained
by procedures used in sequencing, provided the disulfides are not broken prior to cleaving the
polypeptide chain. Because these covalent bonds are stable under most conditions used in the
cleavage of polypeptides, intact disulfides link the peptide fragments containing their specific
cysteinyl residues and thus these linked fragments can be isolated and identified within the
protein digest. An effective way to isolate these fragments is through diagonal
electrophoresis.

A protein digest in which any disulfide bonds remain intact and link their respective
Cys-containing peptides is streaked along the edge of a filter paper and subjected to
electrophoresis. A strip cut from the edge of the paper is then exposed to performic acid
fumes to oxidize any disulfide bridges. Then the paper strip is attached to a new filter paper
so that a second electrophoresis can be run in a direction perpendicular to the first. Peptides
devoid of disulfides experience no mobility change, and thus their pattern of migration
defines a diagonal. Peptides that had disulfides migrate off this diagonal and can be easily

identified, isolated, and sequenced to reveal the location of cysteic acid residues formerly
involved in disulfide bridges. From this information, the positions of the disulfides in the
protein can be stipulated.

MASS SPECTROMETER:
Mass spectrometry (also known as mass spectroscopy informally, "mass-spec" and
MS) is an analytical technique used to measure the mass-to-charge ratio of ions. It is most
generally used to find the composition of a physical sample by generating a mass spectrum
representing the masses of sample components. The mass spectrum is measured by a mass
spectrometer.
For large samples such as biomolecules, molecular masses can be measured to within
an accuracy of 0.01% of the total molecular mass of the sample i.e. within a 4 Daltons (Da)
or atomic mass units (amu) error for a sample of 40,000 Da. This is sufficient to allow minor
mass changes to be detected, e.g. the substitution of one amino acid for another, or a posttranslational modification. For small organic molecules the molecular mass can be measured
to within an accuracy of 5 ppm or less, which is often sufficient to confirm the molecular
formula of a compound, and is also a standard requirement for publication in a chemical
journal. Structural information can be generated using certain types of mass spectrometers,
usually those with multiple analysers which are known as tandem mass spectrometers. This is
achieved by fragmenting the sample inside the instrument and analysing the products
generated. This procedure is useful for the structural elucidation of organic compounds and
for peptide or oligonucleotide sequencing.
APPLICATIONS:
Accurate molecular weight measurements:
Sample confirmation, to determine the purity of a sample, to verify amino acid
substitutions, to detect post-translational modifications, to calculate the number of disulphide
bridges.
Reaction monitoring:
To monitor enzyme reactions, chemical modification and protein digestion.
Amino acid sequencing:
Sequence confirmation, de novo characterisation of peptides, identification of proteins
by database searching with a sequence "tag" from a proteolytic fragment.
Oligonucleotide sequencing:
The characterisation or quality control of oligonucleotides
Protein structure:

Protein folding monitored by H/D exchange, protein-ligand complex formation under

physiological conditions and macromolecular structure determination
COMPONENETS:
Mass spectrometers can be divided into three fundamental parts, namely the ionisation
source , the analyser , and the detector.
The sample has to be introduced into the ionisation source of the instrument. Once
inside the ionisation source, the sample molecules are ionised, because ions are easier to
manipulate than neutral molecules. These ions are extracted into the analyser region of the
mass spectrometer where they are separated according to their mass (m) -to-charge (z) ratios
(m/z). The separated ions are detected and this signal sent to a data system where the m/z
ratios are stored together with their relative abundance for presentation in the format of a m/z
spectrum.
The analyser and detector of the mass spectrometer, and often the ionisation source
too, are maintained under high vacuum to give the ions a reasonable chance of travelling
from one end of the instrument to the other without any hindrance from air molecules. The
entire operation of the mass spectrometer, and often the sample introduction process also, is
under complete data system control on modern mass spectrometers.

IONISATION SOURCE:
The ion source is the part of the mass spectrometer that ionizes the material under
analysis (the analyte). The ions are then transported by magnetic or electric fields to the mass
analyzer. The ionisation methods used for the majority of biochemical analyses are
Electrospray Ionisation (ESI) and Matrix Assisted Laser Desorption Ionisation (MALDI).

MASS ANALYSER:
The main function of the mass analyser is to separate, or resolve, the ions formed in
the ionisation source of the mass spectrometer according to their mass-to-charge (m/z) ratios.
There are a number of mass analysers currently available, the better known of which include
quadrupoles, time-of-flight (TOF) analysers, magnetic sectors, and both Fourier transform
and quadrupole ion traps .
These mass analysers have different features, including the m/z range that can be
covered, the mass accuracy, and the achievable resolution. The compatibility of different
analysers with different ionisation methods varies. For example, all of the analysers listed
above can be used in conjunction with electrospray ionisation, whereas MALDI is not usually
coupled to a quadrupole analyser.
Tandem (MS-MS) mass spectrometers are instruments that have more than one
analyser and so can be used for structural and sequencing studies. Two, three and four
analysers have all been incorporated into commercially available tandem instruments, and the
analysers do not necessarily have to be of the same type, in which case the instrument is a
hybrid one. More popular tandem mass spectrometers include those of the quadrupolequadrupole, magnetic sector-quadrupole , and more recently, the quadrupole-time-of-flight
geometries.
Time of Flight (TOF):
It uses an electric field to accelerate the ions through the same potential, and then
measures the time they take to reach the detector. If the particles all have the same charge,
then their kinetic energies will be identical, and their velocities will depend only on their
masses. Lighter ions will reach the detector first.
Quadrupole:

Quadrupole mass analyzers use oscillating electrical fields to selectively stabilize or

destabilize ions passing through a radio frequency (RF) quadrupole field. A quadrupole mass
analyzer acts as a mass selective filter and is closely related to the Quadrupole ion trap,
particularly the linear quadrupole ion trap except that it operates without trapping the ions. A
common variation of the quadrupole is the triple quadrupole.
DETECTORS:
The detector monitors the ion current, amplifies it and the signal is then transmitted to
the data system where it is recorded in the form of mass spectra. The m/z values of the ions
are plotted against their intensities to show the number of components in the sample, the
molecular mass of each component, and the relative abundance of the various components in
the sample.
The type of detector is supplied to suit the type of analyser; the more common ones
are the photomultiplier, the electron multiplier and the micro-channel plate detectors.
MASS SPECTROMETRY SOFTWARE:
Mass spectrometry software is any software for data acquisition, analysis or data
representation in mass spectrometry. A popular option that combines a comprehensive range
of data analysis features is PEAKS. Other existing mass spec analysis software include:
Peptide fragment fingerprinting (SEQUEST, Mascot, OMSSA and X!Tandem). Peptide de
novo sequencing (LuteFisk, PepNovo, and Sherenga). Peptide sequence tag based searching
(SPIDER, InsPecT, and GutenTAG).
PEAKS:
PEAKS is designed for peptide sequencing and protein identification from tandem
mass spectrometry (MS/MS) data. Other than being used for search engine protein
identification (Protein ID), it is one of the earliest adaptors for de novo sequencing (both
automated and manual) and sequence tag based searching (SPIDER). In short, de novo
sequencing is peptide sequencing performed without prior knowledge of the amino acid
sequence. Some of the information PEAKS provides is a complete sequence for each peptide,
confidence scores on individual amino acid assignments, simple reporting for highthroughput analysis. One of the most useful tools in any form of research is the ability to
compare results. PEAKS inChorus will cross check test results automatically with other
protein ID search engines, like Sequest, OMSSA, X!Tandem and Mascot. This approach
guards against false positive peptide assignments, and provides the highest possible level of
confidence.
SPIDER:
Common database search engines are unable to recognize some peptides. SPIDER, a
sequence tag based search tool, complements protein identification by quickly seeking
homology in proposed protein sequences. Partial sequence recognition allows for a greater
understanding of post translatinoal modifications and sequence mutations.
SEQUEST:

SEQUEST is a popular tandem mass spectrometry data analysis program. Sequest

identifies collections of tandem mass spectra to peptide sequences that have been generated
from databases of protein sequences. This tool is most useful in the context of shotgun
proteomics. Starting with a complex mixture of proteins, this strategy typically employs
trypsin to digest proteins. These peptides are separated by liquid chromatography en route to
a tandem mass spectrometer. The mass spectrometer then isolates ions of a particular peptide,
subjects them to collision-induced dissociation, and records the produced fragments in a
tandem mass spectrum. This process, repeated for several hours, will produce thousands of
tandem mass spectra. Identifying such a data collection requires automation, and Sequest was
the first software to fill that need. Sequest identifies each tandem mass spectrum
individually. The software evaluates protein sequences from a database to compute the list of
peptides that could result from each. The peptide's intact mass is known from the mass
spectrum, and Sequest uses this information to determine the set of candidate peptides
sequences that could meaningfully be compared to the spectrum by including only those
which are near the mass of the observed peptide ion. For each candidate peptide, Sequest
projects a theoretical tandem mass spectrum, and Sequest compares these theoretical spectra
to the observed tandem mass spectrum by the use of cross correlation. The candidate
sequence with the best matching theoretical tandem mass spectrum is reported as the best
identification for this spectrum.

TANDEM MASS SPECTROMETRY

Tandem mass spectrometry (MS-MS) is used to produce structural information about
a compound by fragmenting specific sample ions inside the mass spectrometer and
identifying the resulting fragment ions. This information can then be pieced together to
generate structural information regarding the intact molecule. Tandem mass spectrometry also
enables specific compounds to be detected in complex mixtures on account of their specific
and characteristic fragmentation patterns. A tandem mass spectrometer is a mass
spectrometer that has more than one analyzer, in practice usually two. The two analyzers are
separated by a collision cell into which an inert gas (e.g. argon, xenon) is admitted to collide
with the selected sample ions and bring about their fragmentation. The analyzers can be of
the same or of different types, the most common combinations being:
quadrupole - quadrupole
magnetic sector - quadrupole
magnetic sector - magnetic sector
quadrupole - time-of-flight.
Fragmentation experiments can also be performed on certain single analyzer mass
spectrometers such as ion trap and time-of-flight instruments, the latter type using a postsource decay experiment to effect the fragmentation of sample ions.

Tandem MS (or MS/MS) allows sequencing of proteins by hooking two mass

spectrometers in tandem. The first mass spectrometer is used to separate oligopeptides from a
protein digest and then to select in turn each of these oligopeptides for further analysis. A
selected ionized oligopeptide is directed toward the second mass spectrometer; on the way,
this oligopeptide is fragmented by collision with helium or argon gas molecules, and the
collection of fragments is analyzed by the second mass spectrometer. Fragmentation occurs
primarily in the peptide bonds linking successive amino acids in the oligopeptide, so the
fragments created represent a nested set of peptides that differ in size by one amino acid
residue. The fragments differ in mass by 56 atomic mass units (the mass of the peptide
backbone atoms (NH-CH-CO)) plus the mass of the R group at each position, which ranges
from 1 atomic mass unit (Gly) to 130 (Trp). MS sequencing has the advantages of very high
sensitivity, fast sample processing, and the ability to work with mixtures of proteins.
Subpicomoles (less than 10_12 moles) of peptide can be analyzed. However, in practice,
tandem MS is limited to rather short sequences (no longer than 15 or so amino acid residues).
PEPTIDPEPTIDE SEQUENCING BY TANDEM MASS SPECTROMETRY:
There are three different types of bonds that can fragment along the amino acid
backbone: the NH-CH, CH-CO, and CO-NH bonds. Each bond breakage gives rise to two

species, one neutral and the other one charged, and only the charged species is monitored by
the mass spectrometer. The charge can stay on either of the two fragments depending on the
chemistry and relative proton affinity of the two species. Hence there are six possible
fragment ions for each amino acid residue and these are labelled as in the diagram, with the a,
b, and c" ions having the charge retained on the N-terminal fragment, and the x, y", and z
ions having the charge retained on the C-terminal fragment. The most common cleavage sites
are at the CO-NH bonds which give rise to the b and/or the y" ions. The mass difference
between two adjacent b ions, or y"; ions, is indicative of a particular amino acid residue.
Table of amino acid residues:
Symbol
Ala A
Arg R
Asn N
Asp D
Cys C
Gln Q
Glu E
Gly G
His H
Ile I
Leu
Lys K
Met M
Phe F
Pro P
Ser S
Thr T
Trp W
Tyr Y
Val V

Structure
-NH.CH.(CH3).CO-NH.CH.[(CH2)3.NH.C(NH).NH2].CO-NH.CH.(CH2CONH2).CO-NH.CH.(CH2COOH).CO-NH.CH.(CH2SH).CO-NH.CH.(CH2CH2CONH2).CO-NH.CH.(CH2CH2COOH).CO-NH.CH2.CO-NH.CH.(CH2C3H3N2).CO-NH.CH.[CH.(CH3)CH2.CH3].CO-NH.CH.[CH2CH(CH3)2].CO-NH.CH.[(CH2)4NH2].CO-NH.CH.[(CH2)2.SCH3].CO-NH.CH.(CH2Ph).CO-NH.(CH2)3.CH.CO-NH.CH.(CH2OH).CO-NH.CH.[CH(OH)CH3).CO-NH.CH.[CH2.C8H6N].CO-NH.CH.[(CH2).C6H4.OH].CO-NH.CH.[CH(CH3)2].CO-

Mass (Da)
71.0
156.1
114.0
115.0
103.0
128.1
129.0
57.0
137.1
113.1
113.1
128.1
131.0
147.1
97.1
87.0
101.0
186.1
163.1
99.1

Peptide sequencing by tandem mass spectrometry - backbone cleavages

The extent of side-chain fragmentation detected depends on the type of analyzers used
in the mass spectrometer. A magnetic sector - magnetic sector instrument will give rise to
high energy collisions resulting in many different types of side-chain cleavages. Quadrupole quadrupole and quadrupole - time-of-flight mass spectrometers generate low energy
fragmentations with fewer types of side-chain fragmentations.
Immonium ions (labelled "i") appear in the very low m/z range of the MS-MS
spectrum. Each amino acid residue leads to a diagnostic immonium ion, with the exception of
the two pairs leucine (L) and iso-leucine (I), and lysine (K) and glutamine (Q), which
produce immonium ions with the same m/z ratio, i.e. m/z 86 for I and L, m/z 101 for K and
Q. The immonium ions are useful for detecting and confirming many of the amino acid
residues in a peptide, although no information regarding the position of these amino acid
residues in the peptide sequence can be ascertained from the immonium ions.
An example of an MS/MS daughter or product ion spectrum is illustrated below.

The molecular mass of the peptide was measured using standard mass spectrometric
techniques and found to be 680.4Da, the dominant ions in the MS spectrum being the
protonated molecular ions (M+H+) at m/z 681.4. These ions were selected for transmission
through the first analyzer, then fragmented in the collision cell and their fragments analyzed
by the second analyzer to produce the following MS/MS spectrum. The sequence (amino acid
backbone) ions have been identified, and in this example the peptide fragmented
predominantly at the CO-NH bonds and gave both b and y" ions. (Often either the b series or
the y" series predominates, sometimes to the exclusion of the other). The b series ions have
been labelled with blue vertical lines and the y" series ions have been labelled with red
vertical lines. The mass difference between adjacent members of a series can be calculated
e.g. b3-b2 = 391.21 - 262.16 = 129.05 Da which is equivalent to a glutamine (E) amino acid
residue; and similarly y4 - y3 = 567.37 - 420.27 = 147.10 Da which is equivalent to a
phenylalanine (F) residue. In this way, using either the b series or the y" series, the amino
acid sequence of the peptide can be determined and was found to be NFESGK (note:. the y"
series reads from right to left!). The immonium ions at m/z 102 merely confirm the presence
of the glutamine (E) residue in the peptide.

PEPTIDE MAPPING:
Peptide mapping refers to the comparison of mobilities of peptide fragments from
homologous proteins. Protein sequencing is time consuming process once, the primary

structure of a protein has been elucidated, and however, that of nearly identical protein, e.g.
protein from a closely related species, mutated protein etc., can be more easily determined by
peptide mapping or finger printing. Peptide mapping done by combining paper
chromatography and paper electrophoresis digest. It can also be obtained by single mass
spectrometer. Peptide mass fingerprinting uses the masses of proteolytic peptides as input to
a search of a database of predicted masses that would arise from digestion of a list of known
proteins. If a protein sequence in the reference list gives rise to a significant number of
predicted masses that match the experimental values, there is some evidence that this protein
was present in the original sample.
Peptide fragments incorporating the amino acid
variations migrate to different positions on their peptide map (finger prints). When compared
to the corresponding peptides of the known protein, the sequence of peptide fragments
creating similar spots identified but varying/differing spots are determined by eluting and
sequencing by the normal methods.

REVERSE SEQUENCING:
In this technique, amino acid sequence of protein determined by analyzing the
nucleotide sequence of corresponding DNA that codes for protein. This is because DNA
directs the amino acid sequence in protein. This technique, however, fails to identify the
disulfide bonds and changes that occur in the amino acids after the protein is synthesized i.e.
post translational modifications. Bioinformatic tools like ExPASy Translate tool might be
used for this purpose.