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CALIBRATORS AND INTERNAL STANDARDS IN PROTEIN MS ASSAYS:DIFFERENCES, COMMONALITIES, AND BEST-PRACTICE

CHRISTOPHER M. SHUFORDMSACL-EU, SALZBURG, AUSTRIASEPTEMBER 12, 2018

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PART 1: CALIBRATION (NOT INTERNAL STANDARDIZATION)

WHAT’S YOUR POINT OF VIEW?

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PERSPECTIVE MATTERS

4

RHE

Ag/AgCl

Reference Electrode

INTER-METHOD COMPARISON REQUIRES CALIBRATED RESPONSE

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y = 1.01xR² = 0.97

0

0.5

1

1.5

2

2.5

0 0.5 1 1.5 2 2.5

Rela

tive

SRM

Inte

nsity

, Ins

trum

ent #

2

Relative SRM Intensity, Instrument #1

Reference Sample (i.e., “calibrator")

y = 0.006xR² = 0.97

0

500000

1000000

1500000

2000000

0 500000 1000000 1500000 2000000

Mea

sure

d SR

M In

tens

ity, I

nstr

umen

t #2

Measured SRM Intensity, Instrument #1

Reference Sample (i.e., “calibrator”)

Comparison of results between different methods/instruments requires calibration of response factors

CALIBRATION (X CONCENTRATION = Y RESPONSE)

6

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 1 2 3 4 5 6

Mea

sure

d Re

spon

seM

easu

red

Resp

onse

Analyte (A) Concentration

Calibration defines the response factor, i.e., the relationship between:1) concentration in the original/unprocessed sample and 2) the measured response

Calibrator “known” concentration

Test Sample unknown concentration

RESPONSE FACTORS ARE ASSAY SPECIFIC

7

Protein Enrichment Digestion

Peptide Enrichment

Ionization/ MS1

Fragmentation/ MS2

++

+ +++

+

+

Response Factor is dependent upon process recovery through the assay, not just analytical response

EXTERNAL CALIBRATION

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“known” amounts of

protein

15

1050

External Calibration

Samples

1 5 50

? ? ? ?

??

??

Unknown Test

Samples

10Total Efficiency

Total Efficiency

EXTERNAL CALIBRATION WITH INTERNAL STANDARDIZATION

9

15

1050

External Calibration

Samples

1 5 50

? ? ? ?

??

??

Unknown Test

Samples

10Total Efficiency

Total Efficiency

constant amount of internal standard added to both

calibrator and test samples

INTERNAL CALIBRATION

10

? ? ? ?

??

??

Unknown Test

Samples

Total Efficiency

“known” amount of internal calibrant added to

test samples

CALIBRANT VS INTERNAL STANDARD (IS)

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0

2000

4000

6000

8000

10000

0 2 4 6 8 10

Ana

lyte

Res

pons

e

Analyte Concentration

External Calibration without Internal Standardization

0

0.9

1.8

2.7

3.6

4.5

0 2 4 6 8 10

A:IS

Res

pons

e R

atio

Analyte Concentration

External Calibration with Internal Standardization

(Internal Standard ≠ Calibrant)

Internal Calibration defacto Internal Standardization

(Internal Standard = Calibrant)

0

0.4

0.8

1.2

1.6

2

0 0.4 0.8 1.2 1.6 2

A:IS

Res

pons

e R

atio

A:IS Concentration Ratio

TYPES OF (GOOD & BAD) CALIBRANTS

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Protein1 Protein-LevelEnrichment

2 Cleavable Surrogate

3Peptide

Surrogate

Digestion

Peptide-LevelEnrichment

time

LC-MSDetection

A:IS

C.M. Shuford & D.C. Muddiman, Encyclopedia Anal. Chem. 2013, DOI: 10.1002/9780470027318.a9311

COMPARISON OF DIFFERENT “INTERNAL CALIBRATORS”

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full-length protein

cleavable peptides

tryptic peptides

calibrant

C.M. Shuford & co-workers, Anal. Chem. 2017, 89(14), 7406–7415.

Starting Protein Amount (3 pmol)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

SIL-Tg cSIL tSIL

Calc

ulat

ed P

rote

in A

mou

nt (p

mol

)

Pep 1 Pep 2 Pep 3

ABSOLUTE QUANTIFICATION = HARMONIZATION

14Netzel, B.C. et al., Clin. Chem. 2016, 62 (1), 297-299

LabSurrogate

PeptideCalibrant

CalibratorMatrix

Internal Standard

Mayo FSP BCR®457HumanSerum

peptide

U. Wash. FSPPooled Serum

TgHuman Serum

peptide

ARUP VIFImmunoassay

TgSynthetic cleavable

LabCorp FSPImmunoassay

TgSynthetic cleavable

All full-length Proteins (traceable to BCR®457)

Comparison of 4 Different LC-MS/MS Methods Between 4 Labs

ABSOLUTE QUANTIFICATION = HARMONIZATION

15Netzel, B.C. et al., Clin. Chem. 2016, 62 (1), 297-299

LabSurrogate

PeptideCalibrant

CalibratorMatrix

Internal Standard

Mayo FSP BCR®457HumanSerum

peptide

U. Wash. FSPPooled Serum

TgHuman Serum

peptide

ARUP VIFImmunoassay

TgSynthetic cleavable

LabCorp FSPImmunoassay

TgSynthetic cleavable

0

4

8

12

16

20

24

28

32

36

40

44

0 4 8 12 16

“Lab X” FSPcleavable peptide

Human Serum

cleavable peptide

Example of Bad Calibration

CALIBRATION CONCLUSIONS

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• Calibration enables comparison of quantitative results between different methods and different labs

• Analyte response factors are assay specific (i.e., because of process recovery)

• Calibrant (and calibrators) should reflect the response factor of the endogenous analyte in test samples

• If your analyte is a protein, your calibrant should be a protein

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PART 2: CALIBRATION BEST PRACTICES

ANCHOR YOUR CALIBRATION (TO SOMETHING STABLE)

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Stable AnchoringLess-than-stable Anchoring

LONGITUDINAL MONITORING

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0

20

40

60

80

100

120

140

0 100 200 300

Mea

sure

d Re

sult

Day

Stable Unstable

Calibrator Lot Change

0

20

40

60

80

100

120

140

0 100 200 300

Mea

sure

d Re

sult

Day

Stable Unstable

Calibrator Instability

ESTABLISH TRACEABILITY TO STABLE REFERENCE

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Primary Reference Material

Primary Reference Measurement Procedure

Matrix-based Secondary Reference Material

Secondary Reference Measurement Procedure

Working Assay Calibrator

Laboratory Measurement Procedure

Patient Test Result

See also: Smit et al., J. Proteomics 2014 (DOI: 10.1016/j.jprot.2014.06.015)See also: van den Broek et al., Clin. Chem. 2016 (DOI: 10.1373/clinchem.2015.246702)

Teir 1 Standardize to Reference Method

• Reference Measurement Procedures• National Measurement Institutes • Recognized Reference Labs• Calibrated with Reference Materials

Traceability Chain

ESTABLISH TRACEABILITY TO STABLE REFERENCE

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Primary Reference Material

Primary Reference Measurement Procedure

Matrix-based Secondary Reference Material

Secondary Reference Measurement Procedure

Working Assay Calibrator

Laboratory Measurement Procedure

Patient Test Result

Teir 2 Standardize with Primary Reference Material

• Reference Materials (CRM/SRM; ISO 17511)

• Primary (often recombinant & matrix-free)• Potential non-commutability

(i.e., may not give agreement in test results)

• Secondary (often matrix-based, i.e., pools)• Low chance for non-commutability

Traceability Chain

(NON-)COMMUTABILITY OF REFERENCE MATERIALS

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Both assays calibrated with BCR®457 diluted in serum free of Tg(-)/TgAb(-)

See also: Spencer et al, J. Clin. Endocrinol. Metab. 2005 (DOI: 10.1210/jc.2005-0671)

0

50

100

150

200

250

0 50 100 150 200 250

LC-M

S/M

S [T

g], n

g/m

L

Immunoassay [Tg], ng/mL

Slope bias +36%

HARMONIZATION, THE NEXT BEST THING

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Primary Reference Material

Primary Reference Measurement Procedure

Matrix-based Secondary Reference Material

Comparator Measurement Procedure

Working Assay Calibrator

Laboratory Measurement Procedure

Patient Test Result

Smit et al., J. Proteomics 2014 (DOI: 10.1016/j.jprot.2014.06.015)van den Broek et al., Clin. Chem. 2016 (DOI: 10.1373/clinchem.2015.246702)

Teir 3 Harmonize to Comparator Assay

• Use comparator assay calibrators• Potential non-commutability

(i.e., may not give agreement in test results)

• Use test samples measured by comparator assay• Lower probability of non-commutability• Use test samples (pools?) directly as working

calibrators• Use test samples to assign values of working

calibrators

Traceability Chain

HARMONIZATION

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Both assays calibrated with Immunoassay calibrators

0

50

100

150

200

250

0 50 100 150 200 250

LC-M

S/M

S [T

g], n

g/m

L

Immunoassay [Tg], ng/mL

Slope bias +27%

Nominal Calibrator Values Adjusted Calibrator Values

Adjustment

0

50

100

150

200

250

0 50 100 150 200 250

LC-M

S/M

S [T

g], n

g/m

L

Immunoassay [Tg], ng/mL

Slope bias ~0%

HARMONIZATION, THE NEXT BEST THING

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Primary Reference Material(not always a protein…)

“Alternate” Reference Measurement Procedure

Matrix-based Secondary Reference Material

Secondary Reference Measurement Procedure

Working Assay Calibrator

Laboratory Measurement Procedure

Patient Test Result

Teir 4 Standardize to Higher-order Metrology

• Gravimetrically• Required Purified Material (and a lot of it)• Requires correction for salt/water

• Amino Acid Analysis (AAA) or Nitrogen Analysis• Requires Purified Material• Buffer/Solubility issues

• Spectrophotometry• Requires Purified Material• A210 or 280• Bradford/Lowery Assay

All methods require primary reference standard to ensure stable anchoring (i.e., traceability).

Traceability Chain

Vendor States > 98% Pure

ESTIMATING PROTEIN (IM)PURITY BY SDS-PAGE

Do you believe the Vendor?

• High MW Contaminants• Contaminants >30 kDa?

• Low MW Contaminants?• Contaminants <10 kDa?

• Dynamic Range of Stain• Can you observe 2% impurity?

From Certificate of Analysis

MW (Da)

Your SDS-PAGE ….How pure now? >98%?

ESTIMATING PROTEIN (IM)PURITY BY SDS-PAGE

MW (Da)

Your test …. Continued

~85% Purity – will lead to lot-lot variability

ESTIMATING PROTEIN (IM)PURITY BY SDS-PAGE

MW (Da)

Calibrant ProteinLoad

INTACT MASS (DIS)QUALIFICATION

Courtesy or Dr. Cory Bystrom

[M+6H+]6+

[M+7H+]7+

[M+8H+]8+

[M+9H+]9+

Confirm AA Composition (MS2 for Sequence)

Larger proteins may need bottom-up sequence analysis

Full-Scan (MS1)

Degradation Products

Recombinant isotope labeled Human IGF-1

Improperly folded labeled IGF-1

PROTEIN FOLDING: DOES IT MATTER?

Reference – human IGF-1

Are recombinant proteins good calibrants for endogenous protein analytes?Courtesy or Dr. Cory Bystrom

RESPONSE FACTORS OF NATIVE AND RECOMBINANT PROTEINS

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0.500 1.500 2.500 3.500 4.500 5.500 6.500 7.500 8.500 9.500

0.000

1.000

2.000

3.000

4.000

5.000

0.0

1.0

2.0

3.0

4.0

5.0

sTg cTg rTg CS

DOC

Abs

olut

e Tg

Mea

sure

(pm

ol)

RecombinantProtein

Native HumanProtein

BCR®457(native human)

38 Signature PeptidesRecombinant SIL-Tg Internal Calibrant

13.4% CV 14.2% CV 6.0% CV

C.M. Shuford & co-workers, Anal. Chem. 2017, 89(14), 7406–7415.

HIERARCHY OF CALIBRANTS/CALIBRATORS

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What “behaves” like the native, endogenous protein in the test matrix?

Calibrant

Tier 1: test-matrix pool• 1a: disease/test population• 1b: general population

Tier 2: purified native protein• 2a: CRM/SRM• 2b: non-CRM

Tier 3: purified recombinant protein• 3a – human cell line• 3b – non-human cell line

Analyte-free Matrix

Tier 1: test-matrix pool• 1a: unmodified pool• 1b: depleted pool

Tier 2: animal surrogate matrix• e.g. bovine serum• e.g. chicken serum

Tier 3: stripped/synthetic matrix• e.g. charcoal stripped plasma• e.g. albumin in PBS

SUMMARY

33https://wayback.archive-it.org/7993/20170113121154/http://www.fda.gov/downloads/MedicalDevices/NewsEvents/WorkshopsConferences/UCM499243.pdf

• Calibration enables longitudinal comparisons, only if stable

• Calibration should ideally be traceable to primary reference• If not, harmonize (and continually confirm harmony)• If not, become your own reference method (and use orthogonal checks)

• Choose and qualify your calibrators carefully• Commutability = equivalency of calibrator and test samples• Recombinant proteins are not native proteins

See also: “Selection and Use of Calibrators and Internal Standards for Quantitative Proteomics”FDA Public Workshop: LC-MS in the Clinic; Mark Lowenthal, Ph.D. (NIST)

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PART 3: RESPONSE FACTOR NORMALIZATION(INTERNAL STANDARDIZATION)

EXTERNAL CALIBRATION

35

“known” amounts of

protein

15

1050

External Calibration

Samples

1 5 50

? ? ? ?

??

??

Unknown Test

Samples

10Total Efficiency

Total Efficiency

if Δ[A] = 0,then no bias

EXTERNAL CALIBRATION WITH INTERNAL STANDARDIZATION

36

15

1050

External Calibration

Samples

1 5 50

? ? ? ?

??

??

Unknown Test

Samples

10Total Efficiency

Total Efficiency

constant amount of internal standard added to both

calibrator and test samples

if Δ[A/IS] = 0,then no bias

INTERNAL STANDARDS CAN CORRECT FOR MATRIX EFFECTS

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Protein enrichment

Protein Denaturation

Protein Digestion

Peptide Enrichment

Peptide Derivatization

(LC)MS

PEPTIDE

XXXPEPTIDEXXX

PEPTIDE PEPTIDE PEPTIDE-derivPEPTIDE

XXXPEPTIDEXXX

…XXXPEPTIDEXXX……XXXPEPTIDEXXX…

Source of Matrix Effect

ionizationpeptide derivatizationpeptide enrichment

peptide stabilityprotein denaturation *

peptide digestion *

protein enrichment

…XXXPEPTIDEXXX…XXXPEPTIDEXXX

PEPTIDE

*Full-length Protein ISCleavable Peptide ISNon-cleavable Peptide IS

INTERNAL STANDARDS CAN CONTROL FOR RANDOM VARIANCE

38

0

500

1000

1500

2000

2500

0 10 20

Ana

lyte

Pea

k A

rea

(cou

nts)

Day

0.00

0.15

0.30

0.45

0.60

0.75

0 10 20

A:IS

Pea

k A

rea

Rat

io

Day

0.0

5.6

11.2

16.8

22.4

28.0

0 10 20

Calib

rate

d Co

ncen

trat

ion

Day

CV = 40.5%

Digestion

time

LC-MSDetection

A:IS

SIL Peptide IS

CV = 20.8% CV = 12.5%

Reduced variance from raw response normalization by IS

Reduced variance from daily calibration of digest efficiency

INTERNAL STANDARDS CAN CONTROL FOR PEPTIDE DEGRADATION

39

Ligh

t:H

eavy

Rat

io

Digestion Time (hours)

0 1614121086420

5

10

15

20

25

Peptide IS

Digestion

Peptide IS

D.C. Muddiman & co-workers, Mol. Cell. Proteomics. 2012, 11(9), 7406–7415.

SIL PROTEIN CORRECTS FOR DIGESTION MATRIX EFFECT

40

0%

20%

40%

60%

80%

100%

Full-length Protein IS

cleavable peptide IS

non-cleavable peptide IS

Acc

urac

yPeptide 1 Peptide 2

Calibrator: Recombinant Protein in Chicken SerumTest Sample: Native Protein in Human Serum

C.M. Shuford & co-workers, Anal. Chem. 2017, 89(14), 7406–7415.

INTERNAL STANDARDS MAY NOT WORK

41

0%

20%

40%

60%

80%

100%

IS R

ecov

ery

1X DOC 50X DOC

Calibrators(synthetic matrix)

Test Samples(serum matrix)

Cleavable (Winged) Peptide Internal Standard

accurate?

INTERNAL STANDARDS MAY NOT WORK

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0%

20%

40%

60%

80%

100%

IS R

ecov

ery

1X DOC 50X DOC

Calibrators(synthetic matrix)

Test Samples(serum matrix)

73.2%

82.8%

97.4%95.7% 98.2%101.2%

0%

20%

40%

60%

80%

100%

Serum Pool E (277.7IU/mL TgAb)

Serum Pool G (53.9 IU/mL TgAb)

Serum Pool R (0.0 IU/mL TgAb)

Acc

urac

y

HumanSerum

Calibrator(10 ng/mL)

1:1

1X DOC 50X DOC

Serum #1 Serum #2 Serum #3

Cleavable (Winged) Peptide Internal Standard

inaccurate

accurate

borderline

inac

cura

te

bord

erlin

e

accu

rate

INTERNAL STANDARDS MAY NOT WORK

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0%

20%

40%

60%

80%

100%

IS R

ecov

ery

1X DOC 50X DOC

Calibrators(synthetic matrix)

Test Samples(serum matrix)

73.2%

82.8%

97.4%95.7% 98.2%101.2%

0%

20%

40%

60%

80%

100%

Serum Pool E (277.7IU/mL TgAb)

Serum Pool G (53.9 IU/mL TgAb)

Serum Pool R (0.0 IU/mL TgAb)

Acc

urac

y

HumanSerum

Calibrator(10 ng/mL)

1:1

1X DOC 50X DOC

Serum #1 Serum #2 Serum #3

Cleavable (Winged) Peptide Internal Standard

inaccurate

accurate

borderline

inac

cura

te

bord

erlin

e

accu

rate

(GOOD & BAD) CALIBRANT OPTION

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Intact(top-down)

Digest(bottom-up)

Protein Calibrant

Cleavable SurrogateCalibrant

PeptideSurrogateCalibrant

(GOOD & BAD) INTERNAL STANDARD OPTIONS

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Intact(top-down)

Digest(bottom-up)

Analog IS

PeptideIS

CleavableIS

ProteinIS

ALL OF THE (GOOD & BAD) OPTIONS

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Intact(top-down)

Digest(bottom-up)

Protein Calibrant

Cleavable SurrogateCalibrant

PeptideSurrogateCalibrant

Analog IS

PeptideIS

CleavableIS

ProteinIS

THE ONLY (GOOD) OPTIONS

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Intact(top-down)

Digest(bottom-up)

Protein Calibrant

Analog ISPeptide

ISCleavable

IS

ProteinIS

SUMMARY

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Calibrate a protein assay with a protein(Standardize if possible, otherwise Harmonize)

Internal Standard type is less important (in the absence of matrix effects)

Validate the accuracy of your assay(Parallelism, Spike & Recovery, Mixing Studies, Method Comparison)

©2018 Laboratory Corporation of America All rights reserved. 49

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University of WashingtonAndrew Hoofnagle

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Mayo ClinicBrian NetzelStefan Grebe

MilliporeSigma Corporation*Mitzi Rettinger

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Cerilliant Corp is a subsidiary of MilliporeSigma (formerly Sigma-Aldrich)/Merck KGaA)

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