Amino acids

Biochemistry 3070 – Amino
Acids & Proteins
2
• Proteins are linear copolymers built from monomeric
units called amino acids.
• Twenty amino acids are commonly found in proteins.
• These amino acids contain a variety of different
functional groups:
– Alcohols (R-OH)
– Phenols (Ph-OH)
– Carboxylic acids(R-COOH)
– Thiols (R-SH)
– Amines (R-NH2)
– and others…

Amino Acids
Lecture (1):
 Amino Acids are the building units of proteins. Proteins are polymers of amino
acids linked together by what is called “ Peptide bond”.
 There are about 300 amino acids occur in nature. Only 20 of them occur in
proteins.
Structure of amino acids:
Each amino acid has 4 different groups attached to α- carbon. These 4 groups are :
amino group, COOH gp,
Hydrogen atom and side Chain (R)
R

• At physiological PH (7.4), -COOH gp is dissociated forming a negatively charged
carboxylate ion (COO-) and amino gp is protonated forming positively charged ion
(NH3
+) forming Zwitter ion
Classification of amino acids
I- Chemical classification: According to number of COOH and NH2 groups i.e. according
to net charge on amino acid.
A- Monobasic, monocarboxylic amino acids i.e. neutral or uncharged:
R

5
Biopolymer: the monomeric amino acids are linked through an
amide bond (the carboxylic acids of one AA with the -amino
group of a second)
peptide (< 50 amino acids)
protein (> 50 amino acids)
Peptide or protein (polypeptide)

6
Classification of Amino Acids. AA’s are classified
according to the location of the amino group.
There are 20 genetically encoded-amino acids found in peptides
and proteins
19 are primary amines, 1 (proline) is a secondary amine
19 are “chiral”, 1 (glycine) is achiral; the natural configuration of
the -carbon is L.

8
• “Zwitter” Ions:
• Ions bearing two charges were named zwitter
ions by German scientists; the name still
applies today, especially for amino acids at
neutral pH:
+H3N – CH2 – COO-

9
At low pH, proton concentration [H+]is high.
Therefore, both amines and carboxylic acids
are protonated. (-NH3
+ & -COOH)
At high pH, proton concentration is low.
Therefore, both amines and carboxylic acids
are deprotonated. (-NH2 & -COO-)
At neutral pH, amines are protonated(-NH3
+) and
carboxylates are deprotonated(-COO-)

Biochemistry 3070 – Amino
Acids & Proteins
10
• Even though both acids and amines are present in the same
molecule, they mostly behave as though they were separate
entities:

12
Acid-Base Properties of Amino Acids
Draw the following chemical structures for glycine:
(Non-existent form:) H2N – CH2 - COOH
pH=1: +H3N – CH2 - COOH
pH=7: +H3N – CH2 – COO-
pH=12: H2N – CH2 – COO-

Subclassification of neutral amino acids:
All structures are required
1- Glycine R= H
2- Alanine R= CH3
3- Branched chain amino acids: R is branched such as in:
a - Valine R= isopropyl gp
b- Leucine R= isobutyl gp
c- Isoleucine R = is isobutyl
R is isobutyl in both leucine and isoleucine but branching is different: in leucine →
branching occurs on γ carbon
in isoleucine→ branching occurs on β- carbon
4- Neutral Sulfur containing amino acids:
e.g. Cysteine and Methionine. What is cystin?
5- Neutral, hydroxy amino acids:
e.g. Serine and Threonine

6- Neutral aromatic amino acids:
a- Phenyl alanine : It’s alanine in which one hydrogen of CH3 is substituted with
phenyl group. So it’s called phenyl alanine
b- Tyrosine: - it is P- hydroxy phenyl alanine
- it is classified as phenolic amino acid
c- Tryptophan: as it contains indole ring so it is classified as heterocyclic amino acid
7- Neutral heterocyclic amino acids:
a- Tryptophan: contains indole ring
b- Proline: In proline, amino group enters in the ring formation being α-imino gp so
proline is an α-imino acid rather than α-amino acid

(Lecture 2):
B- Basic amino acids: Contain two or more NH2 groups or nitrogen atoms that act as
base i.e. can bind proton.
At physiological pH, basic amino acids will be positively charged.
e.g.
a- Lysine
b- Arginine: contains guanido group
c- Histidine: is an example on basic heterocyclic amino acids

C- Acidic Amino acids: at physiological pH will carry negative
charge.
e.g. Aspartic acid (aspartate) and Glutamic acid (glutamate). see structures in hand out.
Aspargine and Glutamine: They are amide forms of aspartate and glutamate in which side
chain COOH groups are amidated.
They are classified as neutral amino acids.

II- Classification according to polarity of side chain (R):
A- Polar amino acids: in which R contains polar hydrophilic group so can forms hydrogen bond
with H2O. In those amino acids, R may contain:
1- OH group : as in serine, threonine and tyrosine
2- SH group : as in cysteine
3- amide group: as in glutamine and aspargine
4- NH2 group or nitrogen act as a base (basic amino acids ): as lysine, arginine and histidine
5- COOH group ( acidic amino acids): as aspartic and glutamic .
B- Non polar amino acids:
R is alkyl hydrophobic group which can’t enter in hydrogen bonf formation. 9 amino acids are
non polar ( glycine, alanine, valine, leucine, isoleucine, phenyl alanine, tryptophan, proline and
methionine)

III- Nutritional classification:
1- Essential amino acids: These amino acids can’t be formed in the body and so, it is
essential to be taken in diet. Their deficiency affects growth, health and protein
synthesis.
2- Semiessential amino acids: These are formed in the body but not in sufficient amount
for body requirements especially in children.
Summary of essential and semiessential amino acids:
Villa HM = Ten Thousands Pound
V= valine i= isoleucine l= lysine l= leucine
A = arginine* H= histidine* M= methionine
T= tryptophan Th= threonine P= phenyl alanine
*= arginine and histidine are semiessential
3- Non essential amino acids: These are the rest of amino acids that are formed in the body
in amount enough for adults and children. They are the remaining 10 amino acids.

21
Essential Amino Acids:
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine a
Threonine
Tryptophan a
Valine
Arginine b
Histidine b
a Aromatic b Probably essential

IV- Metabolic classification: according to metabolic or degradation products of amino acids
they may be:
1- Ketogenic amino acids: which give ketone bodies . Lysine and Leucine are the only pure
ketogenic amino acids.
2- Mixed ketogenic and glucogenic amino acids: which give both ketonbodies and
glucose.These are: isoleucine, phenyl alanine, tyrosine and tryptophan.
3- Glucogenic amino acids: Which give glucose. They include the rest of amino acids. These
amino acids by catabolism yields products that enter in glycogen and glucose formation.

Peptides and Proteins
20 amino acids are commonly found in protein.
These 20 amino acids are linked together through “peptide bond forming peptides and
proteins (what’s the difference?).
- The chains containing less than 50 amino acids are called “peptides”, while those
containing greater than 50 amino acids are called “proteins”.
Peptide bond formation:
α-carboxyl group of one amino acid (with side chain R1) forms a covalent
peptide bond with α-amino group of another amino acid ( with the side chain R2) by
removal of a molecule of water. The result is : Dipeptide ( i.e. Two amino acids linked
by one peptide bond). By the same way, the dipeptide can then forms a second
peptide bond with a third amino acid (with side chain R3) to give Tripeptide. Repetition
of this process generates a polypeptide or protein of specific amino acid sequence.

Peptide bond formation:
- Each polypeptide chain starts on the left side by free amino group of the first amino acid
enter in chain formation . It is termed (N- terminus).
- Each polypeptide chain ends on the right side by free COOH group of the last amino acid and
termed (C-terminus).

Examples on Peptides:
1- Dipeptide ( tow amino acids joined by one peptide bond):
Example: Aspartame which acts as sweetening agent being used in replacement of cane
sugar. It is composed of aspartic acid and phenyl alanine.
2- Tripeptides ( 3 amino acids linked by two peptide bonds).
Example: GSH (Glutathione) which is formed from 3 amino acids: glutamic acid,
cysteine and glycine. It helps in absorption of amino acids, protects against hemolysis of
RBC by breaking H2O2 which causes cell damage.
3- octapeptides: (8 amino acids)
Examples: Two hormones; oxytocine and vasopressin (ADH).
4- polypeptides: 10- 50 amino acids: e.g. Insulin hormone

Uncommon Amino Acids
• Hydroxylysine and hydroxyproline, are found in the
collagen and gelatin proteins.
• Thyroxin and 3,3`,5-triiodothyronine, iodinated a.a.
are found in thyroglobulin, a protein produced by the
thyroid gland.
• γ-Carboxyglutamic acid is involved in blood
clotting.
• Finally, N-methylarginine and N-acetyllysine are
found in histone proteins associated with
chromosomes.

Peptide bonds are strong and not broken by conditions that denature proteins, such
as heating.
Prolonged exposure to a strong acid or base at elevated temperatures is required to
hydrolyze these bonds nonenzymically.
Characteristics of the peptide bond
 The peptide bond has a partial double-bond character.
 it is shorter than a single bond, and is rigid and planar .
 This prevents free rotation around the bond between the carbonyl
carbon and the nitrogen of the peptide bond.
 However, the bonds between the α-carbons and the α-amino or α-
carboxyl groups can be freely rotated.
--This allows the polypeptide chain to assume a variety of
possible configurations.

Polarity of the peptide bond
The -C=O and -NH groups of the peptide bond are uncharged, but they are polar and
involved in hydrogen bonding in secondary structure
The C-N distance in a peptide bond is typically 1.32 Å,
which is between the values expected for a C-N single bond (1.49 Å) and a C = N
double bond (1.27 Å) .

Optical Properties of Amino Acids
• The α-carbon of a.a.
is attached to four
different chemical
groups is a chiral or
optically active carbon atom.
• Glycine is the exception.
• amino acids exist in two forms, D and L, that are
mirror images of each other.
• All amino acids found in proteins are of the L-
configuration.

• Amino acids are the building blocks of proteins.
• In the body, they exist as zwitterions.
• Zwitterions can behave as both an acid or a base.

45
Acid-Base Behavior of Amino Acids.
Amino acids exist as a zwitterion: a dipolar ion having both a formal positive and
formal negative charge (overall charge neutral).
Amino acids are amphoteric: they can react as either an acid or a base. Ammonium ion acts as
an acid, the carboxylate as a base.
Isoelectric point (pI): The pH at which the amino acid exists largely in a neutral, zwitterionic
form (influenced by the nature of the sidechain)
pKa ~ 5 pKa ~ 9

46
pI =
pKax + pKay
2
pI =
pKa2 + pKa3
2
pI = 9.7
pI =
pKa1 + pKa3
2
pI = 2.7
pI =
pKa1 + pKa2
2
pI = 6.0

ACIDIC AND BASIC PROPERTIES OF AMINO
ACIDS
• Amino acids in aqueous solution contain weakly
acidic α-carboxyl groups and weakly basic α-amino
groups.
• Each of the acidic and basic amino acids contains an
ionizable group in its side chain.
• Thus, both free and some of the combined amino
acids in peptide linkages can act as buffers.
• The concentration of a weak acid (HA) and its
conjugate base(A-) is described by the Henderson-
Hasselbalch equation.

Derivation of the equation
• For the reaction (HA A- + H+ )
[H+] [A-]
• Ka = ───── ------ (1)
[HA]
• By solving for the [H+] in the above equation,
taking the logarithm of both sides of the equation,
multiplying both sides of the equation by -1, and
substituting pH = -log [H+] and pKa = -log [Ka] we
obtain:
[A-]
• pH = pKa + log ─── ------ (2)
[HA]
It is the (Henderson-Hasselbalch equation)

• Study the acid-base
properties of amino acids.
• Start the titration with the
amino acid in acidic form.
As we slowly increase the
pH we should be able to
plot a graph similar to the
one on the right.
mL NaOH
pH
1
2
3

0
2
4
6
8
10
12
14
0 2 4 6 8 10 12
pH
NaOH Volume, mL
pH vs. NaOH Volume, mL
X
Z
At point Z…?
At point X…?pKa = - log Ka
Ka = X-pKa
Henderson-
Hasselbalch [OH ]
pH pKa log
[H ]


 
HCl + NaOH NaCl + H2O

The titration curve points…
• 1 – where half of the original
acidic amino acid had been
titrated and became a
zwitterion.
• 2 - where the amino acid is
entirely in the zwitterion form.
• 3 – where half of the amino
acid is in the zwitterion form
and half is in the basic form.
1
2
3
Volume NaOH, mL
pH

What does each point mean?
• The pH of the midpoint of the
first leg (1) is the pK value of
the carboxylic acid group.
• The midpoint of the second leg
(2) is known as the isoelectric
point. All the amino acids are in
zwitterion form at this point.
• The pH of the midpoint of the
third leg (3) is equal to the pK of
the –NH3
+
1
2
3
Volume NaOH, mL
pH

Titrate our amino acid solution with NaOH to
see the pH curve as it relates to the amount of
NaOH added.
Use pH meters to monitor pH changes during
our titration.
Construct a graph of pH vs. Volume, mL of
NaOH added.

Physical properties of amino acids:
 Colorless,
 crystals,
 soluble in water,
 insoluble in ether.
 All amino acids( except glycine) are
optically active.
 Amphoteric (react as acidic and basic),
(NH2 and COOH group).
Understanding these physical properties, including charge, solubility
and pKa, aid in designing peptide sequences

Chemical properties of amino acids:
2 reactive groups
– COOH
–NH2
A – COOH Reactions:
1-Ester with alcohol
Amino acids react with alcohol to form ester.

2-With NH3 Amidation.
Aspartic acid ————> Aspargine

3. With Sanger s reagent (1-floro-2,4-di nitro benzene).
Is a test for free amino acid.

3. Methylation and benzoylation: important, detoxification process.

4. With nitrous acids
HNO2 ——> Oxo acid.

Amphoteric properties of amino acids: that is they have both basic and acidic
groups and so can act as base or acid.
Neutral amino acids (monobasic, monocarboxylic) exist in aqueous solution as “
Zwitter ion” i.e. contain both positive and negative charge. Zwitter ion is electrically
neutral and can’t migrate into electric field.
Isoelectric point (IEP) = is the pH at which the zwitter ion is formed. e.g IEP of
alanine is 6
Chemical properties of amino acids:
1- Reactions due to COOH group:
-Salt formation with alkalis, ester formation with alcohols, amide formation with
amines and decarboxylation
-2- Reactions due toNH2 group: deamination and reaction with ninhydrin reagent.
-Ninhydrin reagent reacts with amino group of amino acid yielding blue colored
product. The intensity of blue color indicates quantity of amino acids present.

Ninhydrine can react with imino acids as proline and hydroxy proline but gives yellow
color.
3- Reactions due to side chain (R):
1- Millon reaction: for tyrosine gives red colored mass
2- Rosenheim reaction: for trptophan and gives violet ring.
3- Pauly reaction: for imidazole ring of histidine: gives yellow to reddish product
4- Sakagushi test: for guanido group of arginine andgives red color.
5- Lead sulfide test (sulfur test): for sulfur containing amino acids as cysteine give
brown color.

• Complicated than simply forming amide bonds by mixing the
desired amino acids together in a test tube.
• If solutions containing two amino acids are mixed together,
four different dipeptides (as well as other longer peptides)
will be formed.
(e.g. for a mixture of glycine and alanine the four dipeptides
would be glygly, glyala, alagly, alaala).
• To ensure that only the desired dipeptide is formed the
basic group of one amino acid and the acidic group of the
other must both be made unable to react.
This 'deactivation' is known as the protection of reactive
groups, and a group that is unable to react is spoken of as a
protected group (by attaching to a water-insoluble polymer).
Groups allowed to react – deprotected
- SOLID PHASE PEPTIDE SYNTHESIS

• Peptide chains have two ends, known respectively as the N-terminus
and the C-terminus.
(which end is attached to the polymer depends on the polymer used.)
polyamide beads are used
C-terminus of the peptide is attached to the polymer
The attachment is done by reacting the amino acid with a linkage agent and
then reacting the other end of the linkage agent with the polymer.
Peptide- polyamide link will not be hydrolysed during the subsequent
peptide- forming reactions.
Common linkage agents are di- and tri-substituted benzenes

Linkage agents then join the C-terminus amino acid and resin together as follows:
• The next amino acid also needs to have its amino group
protected to prevent the acids reacting with each other.
• This is done by protecting it with FMOC (9-fluorenylmethoxy-
carbonyl).
(amino acids with side chains of aromatic, acid, basic or highly
polar are likely to be reactive.)

These groups and side chains must also be protected to
prevent unwanted branched chains from forming.
There are four main groups used in this way:
tBu (a tertiary butyl group),
Trt (a triphenylmethyl group),
tBOC (a tertiary butyloxycarbonyl group)
PMC (a 2,2,5,7,8-pentamethylchroman-6-sufonyl group).

• The protected amino acid is then reacted with the amino acid attached to the
polymer to begin building the peptide chain.
• The protection group is now removed from the acid at the end of the chain
(so it can react with the next acid to be added on).
• The new acid is then protected (Step 2) and the cycle continues until a chain of the
required length has been synthesised.
• Once the desired peptide has been made the bond between the first amino acid
and the linkage agent is broken to give the free peptide.

• Aside from the 22 proteinogenic amino acids, there are many
other amino acids that are called nonproteinogenic.
• Those either are not found in proteins
GABA
Hydroxyproline
Selenomethionine
Nonproteinogenic amino acids that are found in proteins
-formed by post translational modification, which is modification
after translation during protein synthesis.
Eg.
The formation of hypusine in the translation initiation factor EIF5A,
through modification of a lysine residue

Such modifications can also determine the localization of the protein,
e.g., the addition of long hydrophobic groups can cause a protein to
bind to a phospholipid membrane.
Some nonproteinogenic amino acids are not found in proteins.
E.g., lanthionine,
2aminoisobutyric acid,
dehydroalanine,
neurotransmitter gammaaminobutyric acid.
-occur as intermediates in the metabolic pathways for standard amino
acids –
E.g., 1. ornithine and citrulline occur in the urea cycle, part of amino
acid catabolism
2. Β amino acid beta alanine (3aminopropanoic acid), which is
used in plants and microorganisms in the synthesis of
pantothenic acid (vitamin B5), a component of coenzyme A.

Amino Acid Analysis (Composition)
• At a low level of resolution, we can determine the amino acid
composition of the protein.
hydrolyze the protein in 6 N HCl, 100oC(undervacuum for various
time intervals)
remove the HCl
the hydrolyzate is applied to an ionexchange or hydrophobic interaction
column
amino acids eluted and quantitated with respect to known standards
Add a non naturally occurring amino acid like norleucine in known
Amounts
(as an internal standard to monitor quantitative recovery during the
reactions)

The separated amino acids are are detected by derivitizing with
ninhydrin or phenyl iso thiocyantate.
The reaction is usually allowed to procedure for 24, 36, and 48 hours
(since amino acids with OH (like ser) are destroyed)
(A time course allows the concentration of Ser at time t=0 to be
extrapolated)
(Trp is also destroyed during the process)
(the amide links in the side chains of Gln and Asn are hydrolyzed to form
Glu and Asp, respectively)

N- and C-Terminal Amino Acid Analysis
• The amino acid composition does not give the sequence of the
protein.
• The N terminus of the protein can be determined by reacting the
protein with fluorodinitrobenzene (FDNB) or dansyl chloride, which
reacts with any free amine in the protein, including the epsilon
amino group of lysine.
• The protein is hydrolyzed in 6 N HCl
the amino acids separated by TLC or HPLC.
Two spots should result if the protein was a single chain, with some
Lys residues.

The labeled amino acid other than Lys is the N terminal amino acid.
The C terminal amino acid can be
determined by addition of carboxypeptidases (cleaves amino acids
from the C terminal)
A time course must be done to see which amino acid is released first
N terminal analysis can also be done as part of sequencing the entire
protein.
Analysis for Specific Amino Acids
Aromatic amino acids can be detected by their characteristic
absorbance profiles.
Amino acids with specific functional groups can be determined by
chemical reactions with specific modifying groups

Sequence in peptides
Two methods exist to determine
the entire sequence of a protein.
1. protein is sequenced.
2. The DNA encoding the protein is sequenced.
(The actually protein can be sequenced by automated, sequential
Edman Degradation)

Edman degradation
In this technique, a protein adsorbed to a solid phase reacts with phenyl
iso thiocyanate
Results an intramolecular cyclization and cleavage of the N-terminal
amino acid (washed from the adsorbed protein and detected by HPLC
analysis)
The yields in this technique are close to 100%.
Hence the maximal length of the peptide which can be sequenced is
about 50 amino acids.

Hence the maximal length of the peptide which can be sequenced is
about 50 amino acids
Endoproteases cleaves protein into peptides
For example,
trypsin cleaves proteins within a chain after Lys and Arg
chymotrypsin cleaves after aromatic amino acids, like Trp, Tyr, and Phe
Chemical cleavage by small molecules can be used as well Cyanogen
bromide, CNBr, cleaves proteins after methionine side chains

1.If the protein contains more than one polypeptide chain, the chains are separated
and purified If disulfide bonds connect two different chains,
the SS bond must be cleaved and each peptide independently purified.
2. Intrachain SS bonds between Cys side chains are cleaved with performic acid.
3. The amino acid composition of each chain is determined
4. The Nterminal and Cterminal residues are identified.
5. Each polypeptide chain is cleaved into smaller fragments, and the amino acid
composition and sequence of each fragment is determined.
6. Step 5 is repeated, using a different cleavage procedure to generate a different and
overlapping set of peptide fragments.
7. The overall amino acid sequence of the protein is reconstructed from the sequences
in overlapping fragments.
8. The position of the SS is located

Peptide profiling
Molecular Mass and Structure Determination Using Mass Spectrometry
to determine the molecular mass and structure of a protein
a molecule is first ionized in an ion source
The charged particles are then accelerated by an electric field into a mass
analyzer where they are subjected to an external magnetic field
The external magnetic field interacts with
the magnetic field arising from the movement of the charged particles, causing
them to deflect
The deflection is proportional to the mass to charge ratio, m/z Ions then enter the
detector which is usually a photomultiplier

Sample introduction into the ion source
• simple diffusion of gases and volatile liquids from a reservoir,
• injection of a liquid sample containing the analyte by spraying a
fine mist,
• (for very large proteins,) desorbing a protein from a matrix using
a laser Analysis of complex mixtures (is done by coupling HPLC
with mass spectrometry in a LCMS).

Ion source:
There are many methods to ionize molecules
• By atmospheric pressure chemical ionization (APCI),
• chemical ionization (CI),
• electron impact (EI).
The most common methods for protein/peptide analyzes
• electrospray ionization (ESI)
• matrix assisted laser desorption ionization (MALDI).

Ionization Techniques
• Electron Impact (EI)
• Chemical Ionization (CI)
• Negative Ion Chemical Ionization (NICI)
• Spray Methods:
• Atmospheric Pressure Chemical Ionization (APCI)
• Fast Atom Bombardment (FAB)
• Field Ionization (FI)
• Good for Gas Phase Samples (e.g. for GC Detector)
• Continuous Ion Source, High degree of
Fragmentation

Ionization Techniques
• Electro spray Ionization (ESI)
• Matrix Assisted Laser Desorption Ionization (MALDI)
• Other Methods:
• Secondary Ion Mass Spectrometry (SIMS)
• Inductively Coupled Plasma (ICP)

• Electrospray:
– An electrical nebulization of liquid that results in the
formation of charged micro droplets
• Electrospray ionization:
– The transfer and ionization of molecules from solution
to gas phase by electrospray

Electrospray: From solution to gas
phase(I)
I. Electrical nebulization of liquid results in the
formation of charged micro droplets.
II. Vaporization increases the charge density on the
surface of the droplets. Electrostatic repulsion
increases.
III. When the electrostatic repulsion exceeds the
surface tension the droplet undergoes coulombic
fission.
IV. The formation of charged ions in the gas phase

Electrical nebulization of liquid and
electrochemical oxidation
Electrochemical oxidation in the metal capillary
(needle) at the positive (+) high voltage
terminal
Electrons
Reduction at (-)
Electrons
High voltage power supply
Ref [1]

Electrospray
Spray of charged
microdroplets

A charged droplet undergoing
coulombic fission
Parent droplet
Offspring droplets
Gomez et al., Phys. Fluids 6 (1994) 404-414

Parent droplet after 1
fission
Vol. = 3.5 m3
Area = 11 m2
Solvent evaporation causes
sequential fissions of charged
droplets
N=51250
R=1.5µm
t=462µs
51250
0.945
43560
0.939
384
0.09
43560
0.848
t=74µs
t=70µs
t=39µs
37026
0.844
326
0.08
37026
0.761
31472
0.756
278
0.07
278
0.03
2
0.003
Asymmetrical fission process:
20 offspring droplets are formed
carrying ~2% of the total mass and
~15% of the net charge.
~20 offspring droplets:
Total volumen = 0.06 m3
Total surface area = 2 m3
The formation of smaller droplets
increases the total surface area and
this relieves the coulombic repulsion
N: No. of charges
R: droplet radius

Electrospray ionization (ESI)
The analyte, dissolved in a volatile solvent ( methanol or acetonitrile),
This analyte is injected through a fine stainless steel capillary at a slow
flow rate into the ion source
A high voltage (34 kV) is maintained on the capillary giving it a positive
charge with respect to the other oppositely charged electrode
The flowing liquid becomes charged with same polarity as the polarity
of the positively charged capillary
The high field leads to the emergence of the sample as a charged
aerosol spray of charged microdrops which reduces electrostatic
repulsions in the liquid

This method essentially uses electrical energy to produce the aerosol
instead of mechanical energy to produce a liquid aerosol, as in the case
of a perfume atomizer Surrounding the capillary is a flowing gas
(nitrogen) which helps to move the aerosol towards the mass analyzer
The microdrops become smaller in size as the volatile solvent
evaporates, increasing the positive charge density on the
drops
Eventually electrostatic repulsions cause
the drops to explode in a series of steps, ultimately
producing analyte devoid of solvent

Electrospray ionization (ESI)
Liquid sample
1-3 kV
needle potential
++
+
+
+
+
++
+
+
++
+
+
+
++
Electrosprayed
‘aerosol’
+
+
+
++
+
+
+
+
Gas-phase ions
Mass spectrometer

Matrix Assisted Laser Desorption
Ionization (MALDI)
 MALDI is achieved in two steps.
• In the first step, the compound to be analyzed is dissolved in a
solvent containing in solution small organic molecules, called
the matrix.
• The second step occurs under vacuum conditions inside the
source of the mass spectrometer.
sample is co-crystallized with a matrix and then irradiated
with laser.
• MALDI provides for the nondestructive vaporization and
ionization of both large and small biomolecules

Introduction
• Mass Spectrometry (MS)
– Vital tool used to characterize and analyze molecules
• Limitations
– Biomolecules and organic macromolecules are fragile
– Molecular ions or meaningful fragments were limited
to only 5-10 kDa at the time
• New technique
– In 1987, Michael Karas and Franz Hillenkamp
successfully demonstrated the use of a matrix to
ionize high molecular weight compounds [1].

MALDI
• Matrix Assisted Laser Desorption/Ionization
(MALDI)
– Method where a laser is used to generate ions of high molecular
weight samples, such as proteins and polymers.
– Analyte is embedded in to crystal matrix
– The presence of an aromatic matrix causes the large molecules
to ionize instead of decomposing.

MALDI
• The mechanism remains
uncertain
• It may involve absorption of
light by the matrix
• Transfer of this energy to
the analyte
– which then ionizes into the
gas phase as a result of the
relatively large amount of
energy absorbed.
– To accelerate the resulting
ions into a flight-tube in
the mass spectrometer
they are subjected to a
high electrical field.

MALDI
MALDI involves
• incorporation of the analyte into a matrix,
(10-6 M solution of the analyte mixed with 0.1 M solution of
the matrix)
• absorption/desorption of laser radiation,
• then ionization of the analyte.
(solvents are then evaporated in a vacuum )

MALDI Matrix
• According to Sigma Aldrich, the matrix must meet
the following properties and requirements [5]:
– Be able to embed and isolate analytes (e.g. by co-
crystallization)
– Be soluble in solvents compatible with analyte
– Be vacuum stable
– Absorb the laser wavelength
– Cause co-desorption of the analyte upon laser
irradiation
– Promote analyte ionization

MALDI Laser
• The MALDI method uses a pulse laser
– Laser fires in intervals
• Pulsed laser produces individual group of ions
– 1st pulse=1st group of ions
– 2nd pulse= 2nd group of ions, etc.
• Each group of ions generated are detected
• With continuous pulsing, the signal resolution
increases

MALDI Advantages
see reference 8
• Gentle Ionization technique
• High molecular weight analyte can be ionized
• Molecule need not be volatile
• Sub-picomole sensitivity easy to obtain
• Wide array of matrices

MALDI Disadvantages
• MALDI matrix cluster ions obscure low m/z
species (<600)
• Analyte must have very low vapor pressure
• Pulsed nature of source limits compatibility
with many mass analyzers
• Coupling MALDI with chromatography can be
difficult
• Analytes that absorb the laser can be
problematic
– Fluorescein-labeled peptides

Amino acids

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