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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)
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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
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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
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“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
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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
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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
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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
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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)
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
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!