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  • Hello and welcome to the next module

  • 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