CALIBRATORS AND INTERNAL STANDARDS IN PROTEIN MS …y = 1.01x R² = 0.97 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 Relative SRM Intensity, Instrument #2 Relative SRM Intensity, Instrument

1

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

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

2

PART 1: CALIBRATION (NOT INTERNAL STANDARDIZATION)

WHAT’S YOUR POINT OF VIEW?

3

PERSPECTIVE MATTERS

4

RHE

Ag/AgCl

Reference Electrode

INTER-METHOD COMPARISON REQUIRES CALIBRATED RESPONSE

5

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

8

“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


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)

11

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

12

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”

13

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


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


LabCorp FSPImmunoassay


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

16

• 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

17

PART 2: CALIBRATION BEST PRACTICES

ANCHOR YOUR CALIBRATION (TO SOMETHING STABLE)

18

Stable AnchoringLess-than-stable Anchoring

LONGITUDINAL MONITORING

19

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

20

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

21







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

22

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

23




Comparator 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

24

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


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


Slope bias ~0%

HARMONIZATION, THE NEXT BEST THING

25

Primary Reference Material(not always a protein…)

“Alternate” Reference 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%?


MW (Da)

Your test …. Continued

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


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

31

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


HIERARCHY OF CALIBRANTS/CALIBRATORS

32

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)

34

PART 3: RESPONSE FACTOR NORMALIZATION(INTERNAL STANDARDIZATION)

EXTERNAL CALIBRATION

35

“known” amounts of

protein

15

1050


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


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

37

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


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?


42

0%

20%

40%

60%

80%

100%

IS R

ecov

ery

1X DOC 50X DOC



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


inaccurate

accurate

borderline

inac

cura

te

bord

erlin

e

accu

rate


43

0%

20%

40%

60%

80%

100%

IS R

ecov

ery

1X DOC 50X DOC



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


inaccurate

accurate

borderline

inac

cura

te

bord

erlin

e

accu

rate

(GOOD & BAD) CALIBRANT OPTION

44

Intact(top-down)

Digest(bottom-up)

Protein Calibrant

Cleavable SurrogateCalibrant

PeptideSurrogateCalibrant

(GOOD & BAD) INTERNAL STANDARD OPTIONS

45

Intact(top-down)

Digest(bottom-up)

Analog IS

PeptideIS

CleavableIS

ProteinIS

ALL OF THE (GOOD & BAD) OPTIONS

46

Intact(top-down)

Digest(bottom-up)

Protein Calibrant

Cleavable SurrogateCalibrant

PeptideSurrogateCalibrant

Analog IS

PeptideIS

CleavableIS

ProteinIS

THE ONLY (GOOD) OPTIONS

47

Intact(top-down)

Digest(bottom-up)

Protein Calibrant

Analog ISPeptide

ISCleavable

IS

ProteinIS

SUMMARY

48

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

Laboratory Corporation of AmericaRussell Grant

Matt CrawfordWill Slade

Pat HollandKyle Cahill

Meghan Bradley

University of WashingtonAndrew Hoofnagle

Rockwood Scientific ConsultingAlan Rockwood

ARUPMark Kushnir

Mayo ClinicBrian NetzelStefan Grebe

MilliporeSigma Corporation*Mitzi Rettinger

Uma SreenivasanJim WaltersKevin Ray

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

Thank You!

Documents

CALIBRATORS AND INTERNAL STANDARDS IN PROTEIN MS …y = 1.01x R² = 0.97 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 Relative SRM Intensity, Instrument #2 Relative SRM Intensity, Instrument