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©2017 Waters Corporation 1 COMPANY CONFIDENTIAL
LC-MS/MS for Bioanalytical Peptide and Protein Quantification: Peptide Level Sample Clean-up
Mary Lame Principal Applications Chemist
©2017 Waters Corporation 2 COMPANY CONFIDENTIAL
Goals of Presentation
Understand value of incorporating mixed-mode SPE into an overall workflow
Answer “Should I purify my tryptic peptides?”
Introduce starting protocols for therapeutic, endogenous, and tryptic peptides
©2017 Waters Corporation 3 COMPANY CONFIDENTIAL
Peptide & Protein Bioanalysis
WORKFLOW
©2017 Waters Corporation 4 COMPANY CONFIDENTIAL
Introduction – Peptide Diversity – Common Sample Prep Techniques
Mixed-mode SPE
Basic SPE Screening Method for Therapeutic and Endogenous Peptides
SPE for Tryptic Peptides
Examples
Outline
©2017 Waters Corporation 5 COMPANY CONFIDENTIAL
Sample Preparation Requirements
Provides maximum analyte recovery Minimizes matrix effects Provides significant increase in sample concentration to meet detection limits Reproducible Straightforward method development Selectively separates peptides from matrix or digest components Fast, ability to run in high throughput format Minimize losses due to non-specific binding Maximize cost:value return
Performance for a diversity of peptides
©2017 Waters Corporation 6 COMPANY CONFIDENTIAL
SPE PEPTIDE CLEAN-UP Therapeutic and Endogenous Peptides
©2017 Waters Corporation 7 COMPANY CONFIDENTIAL
Chemical Properties of Diverse Therapeutic and Endogenous Peptides
Peptide MW pI # of Residues HPLC Index* Octreotide 1019 9.3 8 40.8 Angiotensin II 1046 7.4 8 38.3 Desmopressin 1069 8.6 9 16.8 Vasopressin 1084 9.1 9 7.6 Goserelin 1270 7.3 10 31.7 Angiotensin I 1296 7.5 10 56.2 Somatostatin 1638 10.4 14 52.6 Neurotensin 1673 8.9 13 44.4 Bivalirudin 2180 3.9 20 46.2 BNP 3464 12.0 32 15.9 Teriparatide 4118 9.1 34 90.4 Enfuvirtide 4492 4.1 36 155.9 *higher number = more hydrophobic
©2017 Waters Corporation 8 COMPANY CONFIDENTIAL
Choice of Sample Preparation Technique: Therapeutic and Endogenous Peptides
200 literature articles for therapeutic/endogenous peptide quantification surveyed 2
Most common sample prep techniques identified – Reversed-phase SPE (RP SPE) and protein precipitation (PPT) were very common – Liquid-liquid-extraction (LLE) used in a few cases
Experiments in our labs
– 2 peptides spiked into human plasma at 100 ng/mL – RP SPE, PPT, LLE – Criteria: high analyte recovery (>80%)
low matrix effects (<15%)
2 Van den Broek, I., Sparidans, R., Schellens, J., and Beijnen, J. J. Chromatogr. B, 2008, 872, 1-22.
©2017 Waters Corporation 9 COMPANY CONFIDENTIAL
Current Peptide Sample Preparation Techniques
% Analyte Recovery
Bivalirudin Desmopressin
* < 1% recovery for LLE
% Matrix Effects
0 10 20 30 40 50 60 70 80 90
100
Reversed-phase SPE
PPT LLE -50 -40 -30 -20 -10
0 10 20 30 40 50
* Reversed-phase SPE PPT LLE
©2017 Waters Corporation 10 COMPANY CONFIDENTIAL
Orthogonality: Mixed-mode Ion Exchange and Reversed-phase
©2017 Waters Corporation 11 COMPANY CONFIDENTIAL
Method Development Path to Peptide SPE Screening Protocol
Original
Load Pretreated Sample
Wash 1: 2% Formic acid
Wash 2 or Elute 1: 100% MeOH
Elute 2: 5% NH4OH in 60:40 ACN:MeOH
Optimized
Load: dilute plasma with 4% H3PO4
Wash 1: 5% NH4OH
Wash 2: 20% ACN
Elution: 1% TFA in 75/25 ACN/H2O
Strong Bases: pKa >10
Oasis WCX
Acids: pKa 2-8
Oasis MAX
Strong Bases: pKa >10
Oasis WCX
Acids: pKa 2-8
Oasis MAX
©2017 Waters Corporation 12 COMPANY CONFIDENTIAL
Oasis® PST SPE Protocol for Peptides
PST Protocol
Load: dilute plasma with 4% H3PO4
Wash 1: 5% NH4OH
Wash 2: 20% ACN
Elution: 1% TFA in 75/25 ACN/H2O
Strong Bases: pKa >10
Oasis WCX
Acids: pKa 2-8
Oasis MAX
Dilute with water for RP retention
©2017 Waters Corporation 13 COMPANY CONFIDENTIAL
SPE Recoveries Using Basic Peptide Screening Protocol
0
20
40
60
80
100
120
Oasis MAX Oasis WCX
% S
PE R
ecov
ery
Great results for diverse peptides: Screening protocol results in method for 75% of peptides!
©2017 Waters Corporation 14 COMPANY CONFIDENTIAL
Final SPE Results after BNP, Enfuvirtide, and Somatostatin Methods Optimized
% S
PE R
ecov
ery Minor,
compound specific, modifications for 3 peptides result in excellent recovery for all peptides 0
20
40
60
80
100
120
Screening Protocol Modified Protocol
©2017 Waters Corporation 15 COMPANY CONFIDENTIAL
Sample concentration often required – Improves detection limits
Evaporation of eluates may decrease
peptide recovery due to adsorption
Must meet throughput needs for bioanalysis
Challenges in Peptide Extraction Development
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Up to 15X concentration without evaporation – Concentration often necessary to reach LOD’s with
peptides
Minimizes analyte loss – Minimizes sticking to walls of collection plates – Eliminates problems re-solubilizing after dry-down – Beneficial for thermally unstable peptides
Speed – 96-well plate in <30 min, <20 seconds/sample
Residual Volume – 96-well 1 mL Collection Plate – <15 µl residual volume
SPE Format: Oasis® µElution Plates for Peptide Purification
©2017 Waters Corporation 17 COMPANY CONFIDENTIAL
Final SPE Summary: Therapeutic and Endogenous Peptides
Peptide pI MW % SPE
Recovery % Matrix Effects
Octreotide 9.3 1019 88 <10% Angiotensin II 7.35 1046 82 8% Desmopressin 8.6 1069 104 <11% Vasopressin 9.1 1084 100 -3% Goserelin 7.3 1270 100 -2% Angiotensin I 7.51 1296 109 * Somatostatin 10.4 1638 94 * Neurotensin 8.93 1673 114 6% Bivalirudin 3.87 2180 100 10% BNP 12 3464 84 * Teriparatide 9.1 4118 97 9% Enfuvirtide 4.06 4492 102 *
Maximum recovery = enhanced sensitivity
Minimum matrix
effects = selectivity and
sensitivity
©2017 Waters Corporation 18 COMPANY CONFIDENTIAL
High Sensitivity Peptide Quantification: Desmopressin Dynamic Range 1-20000pg/mL
Xevo TQ-S
1pg/mL
5 pg/mL
Blank human plasma
300 µL human plasma
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High Sensitivity Peptide Quantification Angiotensin II
1 pg/mL
5 pg/mL
Blank plasma
Xevo TQ-S
350 µL human plasma
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SPE PEPTIDE CLEAN-UP Protein Digests
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Digested Protein Bioanalysis: Tandem Quad LC-MS
Identify unique peptides and
transitions WORKFLOW
Protein clean-up (optional)
Peptide clean-up (optional)
Data processing
Protein digestion
LC-MS
Results
Optimize / fine-tune
MS conditions
©2017 Waters Corporation 22 COMPANY CONFIDENTIAL
Peptide Level Clean-up From a Digest
Target Analyte Sensitivity
Interfering Digest Reagents, Buffer and Matrix Salts,
Phospholipids
Achieving Recovery of Target Peptides
High Levels of Unwanted Endogenous
Peptides
Should I clean up my peptides?
Peptides Purified Peptides
Peptide-level clean-up (optional)
System Robustness
©2017 Waters Corporation 23 COMPANY CONFIDENTIAL
Matrix Effects at the Signature Peptide Level Addressing the Problem with Sample Prep
1 nM buffer digest
Blank human serum digest
1 nM in serum digest
~1500 area counts
~500-700 area counts = 2-3X lower!
©2017 Waters Corporation 24 COMPANY CONFIDENTIAL
Mixed-mode Cation Exchange (MCX) and Weak Cation Exchange: Tryptic Peptides
Basic
Strong Basic
Acidic
Strong Acidic
©2017 Waters Corporation 25 COMPANY CONFIDENTIAL
Trypsin specifically cleaves (R) and lysine (K) residues, both basic residues – R pKa 10.15 – K pKa 9.35 – With MCX, the residual positive
charge on the Lys and Arg side chains interact with the sulfonate group
Why Mixed-mode Cation Exchange SPE for Tryptic Peptides?
Peptide Lys Peptide Arg
Tryptic cleavage site
©2017 Waters Corporation 26 COMPANY CONFIDENTIAL For research use only. Not for use in diagnostic procedures
Remove interfering buffer salts and digest reagents Recover unique and generic
signature peptides with high efficiency using a single SPE method Minimize sample loss with
µElution format Concentrate the sample up to
15x
ProteinWorks µElution SPE Kit for Protein Digest Purification
One generic protocol: high recovery for diverse tryptic peptides
020406080
100120
Oasis MCX
©2017 Waters Corporation 27 COMPANY CONFIDENTIAL
Tryptic Peptide SPE Clean-up Cytochrome C GITWGEETLMEYLENPKK
None 5 µg/mL
Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75
%
0
100
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75
%
0
100
17Mar2016_CytochromeC_SPEvsNone_NewColumn_011 4: MRM of 1 Channel ES+ 713.35 > 840.5 (GITWGEETLMEYLENPKK)
2.37e5Area
2.90;6960
17Mar2016_CytochromeC_SPEvsNone_NewColumn_028 4: MRM of 1 Channel ES+ 713.35 > 840.5 (GITWGEETLMEYLENPKK)
2.37e5Area
2.903019
2X> Peak Area SPE
No SPE 3019
6960
©2017 Waters Corporation 28 COMPANY CONFIDENTIAL
2ug/mL - [C-3] - Rep 2
Time4.00 6.00 8.00
%
0
100
4.00 6.00 8.00
%
0
100
30Dec2016_HSA_Urine_SPE_047 F5480.79 > 685.44 (FQNALLVR)
4.59e4Area
01Jan2017_HSA_Urine_047 F5480.79 > 685.44 (FQNALLVR)
4.59e4Area
5.85333
Tryptic Peptide SPE Clean-up Urinary Albumin FQNALLVR
SPE
No SPE
4.5X> Peak Area
Albumin QC overspike
concentration (µg/mL)
©2017 Waters Corporation 29 COMPANY CONFIDENTIAL
Familiarization with starting protocols for therapeutic, endogenous, and tryptic peptides – Providing high recoveries >80% for diverse set of peptides
Highlighted benefits of peptide level clean-up with SPE for tryptic peptides – High recovery for tryptic peptides resulting from digestion of various types of proteins – Purification of tryptic peptides eliminates digest reagents, phospholipids, salts etc, increases
sensitivity and specificity, and improves instrument uptime
Benefits of incorporating mixed-mode SPE for peptide level clean-up – Minimizes matrix effects – Improves assay accuracy and precision – Imparts orthogonality into a bioanalytical workflow
Summary