Post on 06-Mar-2020
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Introduction to Peptides and Proteins for Bioanalysis Using LC-MS
Armand Ngounou Senior Scientist, DMPK
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Peptide and Protein Bioanalysis Workflows
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Goals of Presentation
Background and preparation for LC-MS method development
Understand peptide & protein structure
Familiarity with key terms
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PEPTIDE & PROTEIN STRUCTURE
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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
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Amino Acids Are Connected by a Peptide Bond
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Zwitterions
amino acid ionization state changes w/pH
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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
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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
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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
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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
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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
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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
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Protein Example : Insulin
Images from:https://commons.wikipedia.org
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Protein Examples
IGF-1 CRP
Thyroglobulin Apo lipoprotein A1
Protein images from :https://commons.wikipedia.org
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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
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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)
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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
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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
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PEPTIDE FRAGMENTATION IN MASS SPECTROMETRY
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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
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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
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Product m/z Selection
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Product m/z Selection
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Fragment ions can be predicted using on-line software tools (e.g. Skyline)
Product Ion m/z Prediction Tools
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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
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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
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Digested Protein Bioanalysis: Tandem Quad LC-MS
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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