in the Waters Peptide and Protein Bioanalysis Boot Camp.

My name is Khalid Khan, and I'm part of Health Sciences'

marketing team here at Waters.

Today, I will be presenting on peptide and protein structure.

So let's get started.

Here are a number of workflows for large molecule

biotherapeutic and protein biomolecule analysis.

Today, LC-MS is increasingly used for protein quantification

as an alternative to traditional ligand binding assays.

Proteins can be analyzed by LC-MS,

either using intact protein or surrogate peptide workflows.

Both tandem quadrupole and high resolution mass spectrometers

can be used.

Normal flow and microflow LC systems

are also commonly used with both of these mass spectrometer

systems.

Most of this module will focus on the surrogate peptide

workflow using tandem mass spectrometers,

and understanding your peptides and protein structure

is important when developing both intact and surrogate

peptide workflows.

The areas covered in this presentation

will be the basic structure of amino acids,

peptides, and proteins, including

a few specific examples, such as monoclonal antibodies.

The basic structure of peptides and proteins

has an impact on both the sample treatment and LC-MS method

development.

The ionization and fragmentation of peptides

and how these aspects differ from small molecules

will also be covered.

The presentation is mainly intended

for scientists who already have some experience

in small molecule LC-MS method development.

Their aim is to provide an introduction to peptide

and protein structure and explain

the commonly used terms in peptide protein LC-MS method

development.

The presentation will also prepare you

for subsequent modules in the Waters Peptide and Protein

Bioanalysis Boot Camp.

In this first section, let's look at the structure

of peptides and proteins.

Peptides and proteins are chains of amino acids joined together.

There is no agreed criteria that specifies

the length of an amino acid chain that defines whether it

is called a peptide or protein.

One common definition is that if the amino acid chain consists

of less than 50 amino acids, it is

called a peptide, and more than 50 amino acids,

it is called a protein.

This definition is not absolute, and you

can have large peptides and small proteins

of similar amino acid chain lengths.

All of human proteins are formed from just 20

naturally occurring amino acids, or 21,

if you include selenocysteine.

In terms of molecular weight, peptides

are typically less than 6000 daltons,

whereas proteins can be anywhere from 5800

daltons for a small protein such as insulin

or several hundred thousand daltons for large proteins

such thyroglobulin.

This slide illustrates the mechanism

of how two amino acids join together

to form a peptide bond.

The carboxyl group of one amino acid

reacts with the amine group of another amino acid

to form a peptide bond.

The resultant peptide will have a carboxyl group on one end,

and this is referred to as the C-terminal end.

The amine group is referred to as the N-terminal end.

As we will see later, these peptide bonds

fragment in a highly predictable manner in a mass spectrometer

collision cell.

Amino acids and peptides can exist as zwitterions.

This means that they can have both negative and positive

charges, depending on the pH.

This is an important factor when developing sample clean up

methods at the peptide level.

This will be discussed in more detail in later modules.

The chain of amino acids that form the backbone of a peptide

or protein is referred to as its primary structure.

Amino acids are usually represented by a single letter

or three letter abbreviation.

Here is the table of the 21 amino acids

from which human peptides and proteins are formed.

Some single letters are obvious, for example, G

for glycine and A for alanine.

Others are less obvious, such as K for lysine

and R for arginine.

As we will see later in this presentation,

lysine and arginine are very important when

we discuss the breakdown of large proteins

into smaller peptides using specific enzyme digestion.

This slide illustrates the wide variety of structures

and resultant chemical properties of amino acids.

The chemical structure of the amino acids

influences the polarity, hydrophobicity,

and acidic/basic nature of the resultant peptides

and proteins.

Note that cysteine contains a sulfur atom, which

means that two cysteine amino acids can form

disulfide bonds between them.

These disulfide bonds can form in the same peptide chains

or connect two different peptide chains.

I stated earlier that the diverse properties of peptides

and proteins have a large impact on the sample pretreatment

and LC-MS method development.

Note that some amino acids have a second amine group, which

means that they have multiple sites that

can be protonated to form multiply charged,

positive ions.

As the structures of all amino acids are well known,

it is possible to calculate the mass of a peptide

from its amino acid constituents.

Don't worry you will not have to calculate these manually.

Software tools are available to do this automatically for you.

Software tools, such as Skyline, will automatically

calculate the molecular weight of a peptide

from its amino acid sequence.

For example, the peptide D-E-V-I-L,

which consists of aspartic acid, glutamic acid, valine,

isoleucine, and leucine, will have a mass of 587.31662

daltons.

Note that the table above lists the monoisotopic mass

and average mass.

The monoisotopic mass is the mass where only the most

abundant isotopes are used in the calculation,

i.e., carbon-12, hydrogen-1, oxygen-16.

The average mass has all the minor isotopes

also included in the calculation, i.e., carbon-13,

deuterium, and nitrogen-15.

Proteins can exist in different forms and structures.

So far, we have only discussed the basic amino acid

sequence, which is referred to as the primary structure.

Amino acids can form hydrogen bond interactions

between each other, which influences the shape

of a peptide chain or protein.

The most common structures are a pleated sheet and half a helix.

Bonds and interaction between alpha helices

and pleated sheets result in tertiary structures.

Sulfa bonds between cysteine amino acids

and the peptide chains are common in tertiary structures.

Finally, when more than one different type of peptide chain

is involved, quaternary structure is produced.

This slide illustrates the primary structure

of insulin, which includes two amino acid chains joined

together, the insulin A chain and the insulin B chain.

The diagram on this slide also shows

a diagram of the tertiary structure of insulin.

Here is an example of a peptide drug, desmopressin.

This is a relatively small peptide

comprised of nine amino acids.

LC-MS development of a peptide of this length

can be treated in the same way as a small molecule LC-MS

method.

The peptide can be analyzed directly

by LC-MS and standards that are available for MRM method

development.

One difference from a small molecule ESI mass spectrum

is the presence of a doubly charged positive ion

in addition to the singly charged ion.

This is a key feature of peptide ionization

that will be discussed later in this presentation

and other modules.

Note the doubly charged ion at 535.22

and the singly charged ion at 1069.435.

An example of a small protein is insulin, which

consists of 51 amino acids.

The A chain has 21 amino acids, and the B chain

is 30 amino acids.

The monoisotopic mass of insulin is

5023.6377, which is outside the range

of tandem quadrupole mass spectrometer systems which

typically have a maximum upper range of below 2000 daltons.

However, as insulin forms multiply charged ions

with three, four, and five charges,

it can be analyzed using tandem mass spectrometers.

In this example, the five plus ion is shown at mass 1162.

Insulin also forms three plus and four plus ions.

Note again the disulfide bonds connecting the two amino acid

chains between two existing amino acids.

These are very common protein structures.

Here are some examples of larger proteins,

ranging from insulin like growth factor IGF-1

with the molecular weight of 7649

to thyroglobulin, which has a molecular weight over 660,000

daltons.

The slide also shows medium sized proteins,

such as CRP and apolipoprotein A1, which

have molecular weight in the mid 20,000 dalton range.

We can see that as the size of the proteins

increase, the challenge of measuring the intact protein

gets more difficult and is virtually

impossible using limited range tandem mass spectrometers.

However, we can break down large proteins into smaller peptide

units and analyze these peptides using tandem mass

spectrometers.

This approach is called a surrogate peptide approach

and is widely used in protein bioanalysis and protein

biomarker research.

Antibodies are a specific class of proteins

with a common structure.

They are large Y-shaped proteins with two heavy chains and two

light chains.

The heavy chains are linked to each other by disulfide bonds.

Sulfa bonds also link the light chains with the heavy chains.

Human immunoglobulins and antibody

produce white plasma cells to fight infections.

The heavy chains contains approximately 440 amino acids,

and the light chains contain 220 amino acids.

Monoclonal antibody drugs now form a very important class

of therapeutics and need to be measured in biomedical studies

and clinical research studies.

One of the most widely used monoclonal antibody drugs

today is infliximab, which is used

to treat autoimmune conditions such as Crohn's disease.

Infliximab binds to TNF alpha and has

a molecular weight of approximately 150,000 daltons.

Infliximab is known as a chimeric antibody.

Infliximab binds to TNF alpha.

And infliximab is a chimeric antibody.

So how do we analyze large proteins

of several thousand daltons using tandem quadrupole mass

spectrometers which usually have a limited mass range of less

than 2000 daltons.

The approach used is to break down the proteins

into smaller peptides using digestion with enzymes.

A number of different enzymes are used.

The most commonly used enzyme is trypsin,

which cleaves proteins in very specific locations.

Trypsin cleaves proteins adjacent to lysine

and arginine.

Cleavage is always on the c-terminal side

of the amino acid.

This means that peptides arising from trypsin digestion, which

are called triptych peptides, can

be predicted from the amino acid sequence of the protein.

Online software tools are available to predict

triptych peptides.

These online tools also predict the fragmentation

of those peptides in a mass spectrometer.

This is the basis of the surrogate peptide approach,

where a peptide or peptides are quantified

as a surrogate for the proteins from which

the peptides were derived from.

In some cases, proteins cannot be digested directly by enzymes

such as trypsin and require pretreatment prior

to digestion.

One example of this is treatment of disulfide bonds,

which are reduced and alkylated prior to digestion.

If the amino acid sequence of the triptych peptide

is unique to the protein from which it was derived from,

it is called the signature peptide.