Introduction to Peptides and Proteins for Bioanalysis ......Peptides and proteins are made of amino...

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Introduction to Peptides and Proteins for Bioanalysis Using LC-MS

Armand Ngounou Senior Scientist, DMPK


Peptide and Protein Bioanalysis Workflows


Goals of Presentation

Background and preparation for LC-MS method development

Understand peptide & protein structure

Familiarity with key terms


PEPTIDE & PROTEIN STRUCTURE


Peptides are chains of amino acids (AA) Peptide or Protein

– Peptide: < 50 amino acids – Protein: > 50 amino acids

Typically, peptides are considered as <6000 Da

All proteins and peptides are made from 20 common naturally occurring amino acids

All Peptides/Proteins Are Made of Amino Acids


Amino Acids Are Connected by a Peptide Bond


Zwitterions

amino acid ionization state changes w/pH


Peptides/Proteins Primary Structure

Peptides/Proteins = chain of amino acids Amino acids represented by single letter

or three letter abbreviation

E H R

D

H E

N

G

W K

N D R E


Amino Acids, Symbols, and Abbreviations

Symbol Amino Acid Abbreviation Symbol Amino Acid Abbreviation

G Glycine Gly P Proline Pro

A Alanine Ala V Valine Val

L Leucine Leu I Isoleucine Lle

M Methionine Met C Cysteine Cys

F Phenylalanine Phe Y Tyrosine Tyr

W Tryptophan Trp H Histidine His

K Lysine Lys R Arginine Arg

Q Glutamine Gln N Asparagine Asn

E Glutamic Acid Glu D Aspartic Acid Asp

S Serine Ser T Threonine Thr

U Selenocysteine* Sec

*Cysteine with Sulfur atom replaced by Selenium; sometimes called the 21st AA


Physiochemical Properties

Amino Acid Structure and Properties

Physicochemical of amino acids – Polarity – Hydrophobicity – Acidic or basic

Impact – Protein structure – Function – How proteins behave and are analyzed by

LC-MS


Formulas and Weights of AAs in a Peptide Chain

Each residue is “missing H2O” Software tools are available to calculate

the molecular weight The peptide “DEVIL” would have a mass

of 587.31662 Da

Amino Acid Residue Mass


Primary Structure – Basic amino acid sequence

Secondary Structure – Folding of peptide chain caused by hydrogen bond

interactions

Tertiary Structure – Two or more peptide chains connected by covalent bonds

Quaternary Structure – Interaction of different types of peptide chains and

sometimes other atoms

Protein Structures


A synthetic peptide hormone that controls how the body uses water

Can be analyzed directly by LC-MS Method development is similar to small

molecule process Note doubly charged ion at 535.22 and single

charged ion at 1069.435 – Multiple charged ions are a key feature of

peptide ionization

Peptide Example: Desmopressin

2+ Charged ion

1+ Charged ion


Protein Example : Insulin

Images from:https://commons.wikipedia.org


Protein Examples

IGF-1 CRP

Thyroglobulin Apo lipoprotein A1

Protein images from :https://commons.wikipedia.org


Antibodies are large ‘’Y’’ shaped proteins

Basic antibody structure is the same with four polypeptide chains held together by di-sulfide bonds

Human body produces antibodies to fight infections

Many monoclonal antibody (mAb) drugs have been developed over the last few years

Protein Example: Antibody


mAbs can be engineered as therapeutics

Infliximab is a mAb therapeutic used to treat autoimmune diseases – Designed to bind to TNF alpha – Molecular weight ~150 kDaltons – Chimeric antibody

Monoclonal Antibody Drugs (mAbs)


Proteolysis…Proteases/enzymes

Most commonly used – Trypsin C-term to Lys (K), Arg(R)

pH 8.5 – Chymotrypsin C-term to Y, F, W, H, L

pH 8.5 – Arg-C C-term to Arg

pH 8 – Asp-N N-term to Asp

pH 8 – Lys-C C-term to Lys

pH 8.5

E H

R D

H

E N

G

W D

N

K H Q


Proteolysis…Proteases/enzymes

Unique ‘’Signature Peptide’’ – DHENGWDNK

Tryptic peptides should contain 8 and 20 amino acids

Avoid amino acids that can be chemically modified – Cysteine, Methionine

E H

R

D

H E

N

G

W D

N K H Q


PEPTIDE FRAGMENTATION IN MASS SPECTROMETRY


Peptides form multiply charged ions – 2+, 3+, 4+ charged peptide ions

Peptide fragments generated in MS collision cell will have fewer charges than precursor ions

Peptides fragment in highly predictable manner

LC-MS Analysis of Proteins and Peptides


Peptide Fragmentation in Mass Spectrometry

Peptide bond cleavage in CID mode (y and b ions)

Example MS/MS spectra for the peptide : ITDSNAFVLAVK

y and b ions are used in MRM analysis


Product m/z Selection

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Product m/z Selection


Fragment ions can be predicted using on-line software tools (e.g. Skyline)

Product Ion m/z Prediction Tools


Peptide Fragmentation Summary

Peptides form multiply charged ions Peptides fragment in a highly predictable

manner in a mass spectrometer Peptide ion fragments can be predicted using

software tools Resultant y ions often have with higher m/z

than precursor m/z MRM transitions are evaluated and selected

based on specificity and intensity


Peptides and proteins are made of amino acids and can form a variety of complex structures

Small proteins and peptides can be analyzed directly (intact) by tandem quadrupole LC-MS

Larger proteins usually require digestion to smaller peptides for quantification by tandem quad LC-MS

Enzymatic cleavage sites are predictable and software tools are available that can predict tryptic peptides

The structure of peptides and proteins impacts all stages of the bioanalysis workflow

Key Summary Points


Digested Protein Bioanalysis: Tandem Quad LC-MS


Resources

Repositories – Peptide Atlas: www.peptideatlas.org – SRM Atlas: www.srmatlas.org – Pride Archive: www.ebi.ac.uk/pride/archive – Global Proteome Machine:

http://www.thegpm.org/

Open source MRM analysis tools – Skyline

www.skyline.gs.washington.edu/labkey/project/home/begin.view?

Open source MRM design tools – MRMaid: www.138.250.31.29/mrmaid – Seattle Proteome Center:

www.tools.proteomecenter.org/wiki/index.php?title=Software:TPP-MaRiMba

NIH – BLAST:

www.blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome

http://www.peptideatlas.org/

http://www.srmatlas.org/

http://www.ebi.ac.uk/pride/archive

http://www.thegpm.org/

https://skyline.gs.washington.edu/labkey/project/home/begin.view?

https://skyline.gs.washington.edu/labkey/project/home/begin.view

http://138.250.31.29/mrmaid

http://www.tools.proteomecenter.org/wiki/index.php?title=Software:TPP-MaRiMba

http://www.blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome

Introduction to Peptides and Proteins for Bioanalysis ......Peptides and proteins are made of amino...

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