tools and workflows to simplify method development for targeted … · 2016-11-23 · ©2016 waters...

©2016 Waters Corporation 1

Tools and workflows to simplify method development for targeted

MRM methods

Nikunj Tanna Senior Scientist, Scientific Operations

Waters Corporation, Milford, MA


Journey of a large molecule Protein in

Plasma/Serum

LC-MS Data

Protein-level clean-up (optional)

Peptide-level clean-up

(optional)

Peptides Digestion

Purified Protein

Purified Peptides

Pick unique peptides & transitions

Optimize/Fine-tune MRM transitions


•BLASTp •SIM

•Protein Prospector •Skyline

•ProteinWorks •SPE

•MassLynx


ANALYTE

Biomarker

ADC

Modified Peptides/ Proteins

Therapeutic mAb

• Whole Protein? • Sub-unit?

• Human/pre-clinical? • HC Isotype? • CDR sequence?

• Sequence change? • AA modifications?

• Total Ab? • Conjugated Ab? • Payload/Linker?


Tools used

• NCBI BLASTp – Uniqueness against proteome of interest

• SIM – Protein sequence alignment tool


Find the most similar sequences of LC/HC • Protein BLAST against the NR NCBI database

Align the therapeutic LC/HC sequence with the common mAb sequence • SIM (alignment tool from ExPASy Proteomics server)

Generate tryptic peptides while maintaining the sequence alignment of LC/HC •Visually identify possible unique peptides

Find how unique are the un-matched tryptic peptides from therapeutic mAb • Protein BLAST against the NCBI database

Workflow for finding unique tryptic peptides from therapeutic mAbs


Trastuzumab BLAST Results

Trastuzumab LC sequence


Trastuzumab LC vs IgG1kappa 1

mouse IgG1 LC peptide

proteotypic peptide deamidation site

OK

too long (36 AA)

monkey peptide mouse peptide human/mouse peptide human/lama peptide bacterial

peptide OK OK

human/lama peptide

OK OK monkey/human peptide OK


Peptide Level Blast: IYPTNGYTR HC


FINAL LIST of unique peptides

Light Chain – ASQDVNTAVAWYQQK (mouse

peptide)

– LLIYSASFLYSGVPSR – FSGSR

Heavy Chain – EVQLVESGGGLVQPGGSLR (monkey

peptide) – LSCAASGFNIK (mouse peptide, has

Cys) – DTYIHWVAR – QAPGK (human/Llama peptide) – GLEWVAR (human/mouse peptide) – IYPTNGYTR (bacterial peptide) – YADSVK (human/Llama peptide) – FTISADSK – NTAYLQMNSLR (has Met) – AEDTAVYYCSR (human/Llama

peptide, has Cys) – WGGDGFYAMDYWGQGTLVTVSSASTK

(has Met and is too long: 26 AA) – EEMTK (too short: 5 AA, missed

cleavage)


Tools used

• Protein Prospector – Precursor/product mass prediction • MassLynx – PIC, Cone Voltage and Collision Energy optimization • Skyline


Found and optimized Precursor

MRM Fragments

Protein Prospector prediction - FTISADTSK


100 nM trastuzumab digest – Product scan

y5 y6 y7 y8 0

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

10 20 30 40 50 60

CV (V)

Peak area

485.2->721.4

485.2->608.3

485.2->822.5

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

15 20 25 30 35 40 45 50

CE (eV)

Peak area

485.2->721.4 485.2->608.3

485.2->822.5

CE formula = 0.034 m/z +3.1

Fixed CV = 35 V

MassLynx Optimization - FTISADTSK


Skyline Workflow

Target/Analyte sequence and matrix information

In-silico digestion, peptide selection, transition prediction

Pick best 5 MRM transitions from DDA/MSe data

CE Optimization

Final MRM Method

PrecursorPrecursor scan. Pick best precursor

Generate base MRM method with calculated CE

Pick best 5 MRM transitions from MRM data

CE Optimization

Final MRM Method

HRMS Data No HRMS Data

Acquisition & Data Review 1







SkyLine

In silco generation of MRM transitions for peptides

Output scouting MRM method

MassLynx/Xevo TQ-XS

Acquisition of MRM traces for all proposed transitions

SkyLine

Comparison of overlaid MRM traces, to pick optimal transitions &

collision energies

Output final MRM method

MassLynx/Xevo TQ-XS

Sample analysis

SkyLine is developed by the MacCoss laboratory in the University of Washington

Skyline Workflow


MRM Method Creation

With Skyline, importing a protein sequence elicits an in silico digestion algorithm and MRM transition prediction


MRM Method Creation


Sample Analysis with MassLynx


MRM Method Optimization Xevo TQ-XS

Transitions are optimized by collision energy and the results are visualized

The optimized method can be exported automatically


• ProteinWorks Kits

• Protein level clean up (depletion, generic affinity, specific affinity)?

• Peptide level clean up (SPE)?

Sample Preparation Optimization


ProteinWorks Kits in the Workflow Protein in

Plasma/Serum

LC-MS Data

Protein-level clean-up (optional)

Peptide-level clean-up

(optional)

Peptides Digestion

Purified Protein

Purified Peptides

eXpress Digest Kit •Denaturation •Reduction •Alkylation •Enzymatic Digestion •Quench

µElution SPE Kit Target Peptide

Clean-up

Pick unique peptides & transitions

Optimize/Fine-tune MRM transitions


Kit Flexibility: A Diversity of Proteins

Cytochrome C Small Protein MW 12,327 35 µL sample 10 min digestion

Apolipoprotein a1 Biomarker MW 28,300 15 µL sample 15-30 min digestion

Infliximab Biotherapeutic MW 149,100 35 µL sample 2 hour digestion

ProteinWorks Achieves Fast Digestion Times


Flexibility: Across different mAbs & multiple plasma volumes

Protein Peptide Linear fit (r2) with 1/x weighting Mean % Accuracy

Std. Curve Range Standard Curve Range 5.0-50.0 (µg/mL)

15 µL plasma

35 µL plasma

70 µL plasma

15 µL plasma

35 µL plasma

70 plas

Infliximab SINSATHYAESVK 0.999 0.999 0.997 100.00 99.99 99.

DILLTQSPAILSVSPGER 0.999 0.998 0.994 99.99 100.00 100

Trastuzumab

FTISADTSK 0.997 0.993 0.998 100.00 100.01 99.

DTYIHWVR 0.995 0.995 0.996 100.00 100.02 100

IYPTNGYTR 0.998 0.996 0.991 99.98 99.98 98.

Bevacizumab STAYLQMNSLR 0.999 0.998 0.995 100.00 100.01 99.

FTFSLDTSK 0.999 0.999 0.993 100.02 100.00 100

Adalimumab APYTFGQGTK 0.994 0.997 0.995 99.99 99.99 99.

NYLAWYQQKPGK 0.997 0.998 0.999 99.99 100.02 100

Linear, precise and accurate with multiple plasma volumes for multiple mAbs


Calibration curves and QC’s


0.5-500 µg/mL

Calibration curves and QC’s


Thank you!

Acknowledgements

• Mary Lame • Paula Orens • Erin Chambers • Mark Wrona • Kerri Smith • Yun Alelyunas • Kelly Doering Useful insights from • Kevin Bateman (Merck) • Dan Spellman (Merck) Skyline Development Team (University of Washington)

tools and workflows to simplify method development for targeted … · 2016-11-23 · ©2016 waters...

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