lc-ms/ms for bioanalytical peptide and protein quantification · ©2017 waters corporation company...

©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


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


Peptide & Protein Bioanalysis

WORKFLOW


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


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


SPE PEPTIDE CLEAN-UP Therapeutic and Endogenous Peptides


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


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.


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


Orthogonality: Mixed-mode Ion Exchange and Reversed-phase


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


Oasis WCX

Acids: pKa 2-8

Oasis MAX


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


Oasis WCX

Acids: pKa 2-8

Oasis MAX

Dilute with water for RP retention


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!


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


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


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


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


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


High Sensitivity Peptide Quantification Angiotensin II

1 pg/mL

5 pg/mL

Blank plasma

Xevo TQ-S

350 µL human plasma


SPE PEPTIDE CLEAN-UP Protein Digests


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


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


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!


Mixed-mode Cation Exchange (MCX) and Weak Cation Exchange: Tryptic Peptides

Basic

Strong Basic

Acidic

Strong Acidic


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


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


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)


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

lc-ms/ms for bioanalytical peptide and protein quantification · ©2017 waters corporation company...

Documents