1
There Are No Absolutes, Particularly in Protein Quantification 1. Center for Esoteric Testing, Laboratory Corporation of America® Holdings, Burlington, North Carolina, 27215 2. MilliporeSigma, St. Louis, Missouri, 63103 3. MilliporeSigma, Round Rock, Texas, 78665 Introduction Quantitative bottom-up proteomics has been termed “accurate” when using stable isotope-labeled (SIL) surrogates of the protein as a calibrator (i.e., internal calibration). These SIL materials are added as a peptide pre- or more conventionally post-digestion, as a cleavable (winged) peptide pre-digestion or as an intact proteoform prior to denaturation and digestion. This technique differs from classical externally calibrated, internally standardized (normalizing) assay workflows and forms the basis of this paper - is either technique accurate? Fully-tryptic SIL peptides, cleavable SIL peptides, and a full-length SIL protein were compared as internal calibrants and internal (normalizing) standards for quantifying 3 forms of unlabeled thyroglobulin (Tg) by protein cleavage-isotope dilution mass spectrometry using multiple digestion conditions. All SIL materials and unlabeled proteins were standardized by amino acid analysis (NIST traceable) for assignment of accuracy. Methods Figure 1. Eight fully-tryptic SIL peptides (tSIL), 8 cleavable SIL peptides (cSIL), and SIL-rTg were compared as internal calibrators and as internal (normalizing) standards for quantifying 3 samples of unlabeled Tg. The 3 samples of unlabeled Tg quantified (in 0.1% human serum albumin, HSA) were BCR®457 (sTg, thyroglobulin international reference material [1]), a commercially available purified human thyroglobulin (cTg), and a full- length recombinant protein (rTg) expressed and purified in identical fashion to the full-length SIL-rTg, but without labeled amino acids. All SIL materials were labeled at different amino acid positions within the final signature peptide sequences to ensure selective LC-MS/MS analysis of each peptide-form in the same sample. Purity of neat stock solutions of each SIL material and unlabeled Tg proteins were qualified by HPLC (peptides) or SDS- PAGE (proteins) and then standardized by triplicate amino acid analysis (AAA) using NIST amino acid calibration materials (CV<5%). A NIST-traceable BSA control was run in parallel to qualify the accuracy of the AAA (Bias <2%, CV<5%). Internal Calibration versus Internal Standardization Absolute Digestion Efficiency Uncontrolled Digestion-based Matrix Effects References Patricia L. Holland , 1 Christopher M. Shuford, 1 James J. Walters, 2 Kevin Ray, 2 Uma Sreenivasan, 3 Sarah Aijaz, 3 Russell P. Grant 1 1. Feldt-Rasmussen, U. et al. Purification and Certification of Human Thyroglobulin Reference Material CRM457 Final Report; EUR 15611 EN; European Commission Community Bureau of Reference: Luxembourg, Brussels, 1994. 2. Konopka, A.; Boehm, M. E.; Rohmer, M.; Baeumlisberger, D.; Karas, M.; Lehmann, W. D. Anal. Bioanal. Chem. 2012, 404 (4), 1079–1087. 3. Heudi, O.; Barteau, S.; Zimmer, D.; Schmidt, J.; Bill, K.; Lehmann, N.; Bauer, C.; Kretz, O. Anal. Chem. 2008, 80 (11), 4200–4207. Figure 2. All working solutions were gravimetrically prepared to enable traceability to the original AAA-assigned values. For quantification, samples were prepared gravimetrically, then denatured and digested prior to LC- MS/MS analysis. SIL-rTg was added to samples prior to denaturation, cSIL peptides (purity corrected) prior to digestion, and tSIL peptides (purity corrected) after digestion. Additionally, 3 different denaturing conditions (plus DTT and heating) were tested including; 4 M urea (1M during digestion), 1% sodium deoxycholate (DOC, 0.25% during digestion), and 10% trifluoroethanol (TFE, 2.5% during digestion). For internal calibration, quantities of Tg were calculated by taking the quotient of the unlabeled:SIL peptide peak areas measured and multiplying by the absolute amount of the SIL calibrator added. Conclusions Recombinant versus Native Proteins Peptide Internal Calibrators (Tryptic or Cleavable) Do NOT facilitate absolute quantification due to < 100% digestion efficiency of native Tg Do NOT facilitate inter-assay agreement due to variability in digestion efficiency (not commutable) (Even small) impurities in cleavable peptide calibrators will confound standardization Peptide Internal Standards (Tryptic or Cleavable) Require external protein calibrators to achieve accuracy (matrix equivalency for calibration and sample) Purity (concentration) is less important (requires no contribution to/from Native proteoforms) Protein Internal Calibrators Recombinant proteins are surrogate calibrators (at least the big ones) Commutability not guaranteed (peptide, digestion, and IS dependent) Only feasible method for accurate internal calibration (requires harmonization/response balancing of recombinant SIL protein and may not agree with native protein) Protein Internal Standards Recombinant proteins are analogue internal standards (at least the big ones) Only form of IS likely to normalize for digestion-based matrix effects (confounded by PTM’s and native protein binding partners) Only form of IS able to normalize for protein-enrichment variance (confounded by PTM’s and native protein binding partners) Figure 3. (a-c) Addition of SIL materials as internal calibrators demonstrated bias > ±20% for the majority of peptides and denaturing conditions using tryptic peptides (tSIL). Cleavable (cSIL) internal calibrators yielded increased recoveries, with positive bias resulting from differential yield of labeled versus native peptide. Recombinant labeled protein (SIL-rTg) as an internal calibrator provided superior results, however, systematic bias versus all native forms was observed – even compared to recombinant native protein supporting the hypothesis that labeled materials can digest differentially even when expressed identically (see rTg versus SIL-rTg [2]). (d-f) External calibration using unlabeled rTg with SIL materials added as internal standards (I’sS) reduced measurement bias irrespective of the SIL IS added, albeit, certain peptides (TFP) were not fully corrected. Figure 4. Internal calibration using tSILs (Figure 3a) demonstrates that the resulting quantity is the amount of peptide product produced via digestion rather than the starting amount of protein substrate. The digestion efficiency of the protein or cleavable substrate can thus be determined by comparing the amount of peptide product (C peptide , calculated using tSIL calibrators) to the absolute amount of substrate added (C Substrate ) to elucidate sources of bias. = × 100 The source of bias in Figure 3 is derived by the digestion efficiency differences between the unlabeled protein analytes and surrogate calibrants. Using the GGA peptide as an example, the GGA cSIL peptide demonstrates equivalent digestion efficiency versus native protein forms using DOC and urea (Figure 4) – thus, resulting in no bias (Figure 3b); however, increased digestion efficiency was observed for the cSIL versus all 3 unlabeled proteins using trifluoroethanol resulting in positive bias in the accuracy determination (Figure 3b). Figure 6. Disparity between the recombinant and human-derived Tg could arise through differences in the PTM state of the 39 signature peptides rather than differences in digestion efficiency. Determination of the differences in glycosylation were evaluated (total glycan composition, not positional glycosylation). Released glycans (a) MAN5; b) MAN6; c) G2F; d) MAN8; e) MAN9; f) G2FS; g) FA3S) from both the sTg (Left inset) and rTg (Right inset) demonstrate glycosylation of rTg with a modified profile (versus sTg), potentially impacting tertiary and quaternary structure together with digestion efficiency. Figure 7. Digestion variance in potential calibrator matrices for human serum was studied. Surrogate matrices spiked with rTg served as external calibrators, while “unknown” samples were prepared by spiking 100 fmol sTg or cTg into Tg-deficient human serum. The three SIL materials (tSIL, cSIL, and SIL-rTg) were used as internal standards for the FSP (top) and VIF (bottom) signature peptides. Results demonstrate using either human- derived HSA or recombinant HSA (rHSA) as surrogate matrices generated acceptable recoveries regardless of the internal standard or digestion condition. A matrix effect was observed between human and chicken serum for the FSP signature peptide (and not the VIF peptide) with DOC as the denaturant; resulting in negatively biased quantification (≤80% recovery) of both sTg and cTg in human serum using the cSIL or tSIL ISs. Modification of the denaturant conditions ameliorated this bias to some degree indicating the matrix effect is dependent on the combination of digestion condition, signature peptide, and surrogate matrix selected. tSIL Peptides cSIL Peptide Sequence Purity Sequence Purity FSPDDSAGASALLR 94.0% FYQRRRFSPDDSAGASA L LR SGPYMP 78.1% LEDIPVASLPDLHDIER 98.5% VTWKSRLEDIPVASLPD L HDIER ALVGKD 59.7% TFPAETIR 97.6% LHLDSKTFPAET I R FLQGDH 67.3% VIFDANAPVAVR 97.0% KVPESKV I FDANAPVAVR SKVPDS 94.4% VILEDK 100.0% ALFRKKVI L EDK VKNFYT 92.0% SQAIQVGTSWK 88.2% GRLLGRSQA I QVGTSWK QVDQFL 92.1% GGADVASIHLLTAR 86.4% SLAADRGGADVASIH L LTAR ATNSQL 91.4% EFSELLPNR 96.6% GGENYKEFSE L LPNR QGLKKA 99.5% [U- 15 N, 13 C]-labeled residues 30 min, 56 °C 10 mM DTT +Denaturant cleavable SIL (cSIL) peptide unlabeld Tg SIL-rTg LC-SRM 1.3 1.4 1.5 1.6 1.7 Retention Time (min) fully-tryptic SIL (tSIL) peptide tSIL cSIL 3 pmol Full-length (unlabeled Tg) Full-length (SIL-rTg) 3 pmol 3 pmol (each) 1.5 pmol(each) 0.1% HSA 30 min, 37 °C 1:10 Substrate:Enzyme TPCK-treated Trypsin 0.0 1.0 2.0 3.0 4.0 5.0 6.0 sTg cTg rTg 0.0 1.0 2.0 3.0 4.0 5.0 6.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 DOC UREA TFE DOC UREA TFE DOC UREA TFE DOC UREA TFE DOC UREA TFE DOC UREA TFE DOC UREA TFE DOC UREA TFE Absolute Tg Measure (pmol) 0.0 1.0 2.0 3.0 4.0 5.0 6.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 DOC UREA TFE DOC UREA TFE DOC UREA TFE DOC UREA TFE DOC UREA TFE DOC UREA TFE DOC UREA TFE DOC UREA TFE sTg cTg Absolute Tg Measure (pmol) a b c d e f FSP LED TFP VIF VIL SQA GGA EFS FSP LED TFP VIF VIL SQA GGA EFS DOC UREA TFE DOC UREA TFE DOC UREA TFE DOC UREA TFE DOC UREA TFE DOC UREA TFE 0% 20% 40% 60% 80% 100% 120% DOC UREA TFE Digestion Efficiency 0% 20% 40% 60% 80% 100% 120% DOC UREA TFE Digestion Efficiency Absolute Digestion Efficiency EFS VIL SQA GGA VIF FSP LED TFP SIL-rTg cSILs sTg cTg rTg Figure 5. Additional quantification of BCR®457 (sTg) and catalog human-derived Tg (cTg) was determined using 39 peptides with recombinant (rTg) as an external calibrator and SIL-rTg as the internal standard. The reduced variance between peptides (CV = 5.7 – 7.5%) for rTg using SIL-rTg is due to identical digestion efficiencies between unlabeled and SIL-rTg (i.e., common recombinant expression), akin to the analysis of biotherapeutics [3]. Increased variance was observed for the two human-derived Tg materials (sTg CV = 13.0 – 19.4%, cTg CV = 13.8 – 17.9%), explained by disparity in the digestion efficiency of some signature peptides relative to rTg and SIL-rTg, resulting in quantitative bias for those uncontrolled signature peptides. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 DOC UREA TFE Absolute Tg Measure (pmol) sTg cTg rTg sTg cTg rTg sTg cTg rTg Relative Intensity 100 80 60 40 20 sTg a b c d e f g 15 25 35 45 55 20 30 40 50 60 Retention Time (min) rTg a b c d e f g 15 25 35 45 55 20 30 40 50 60 Retention Time (min) 0 0 20 40 60 80 100 120 0 20 40 60 80 100 120 DOC UREA TFE DOC UREA TFE DOC UREA TFE Chicken Serum 6% HSA 6% rHSA 0 20 40 60 80 100 120 DOC UREA TFE DOC UREA TFE DOC UREA TFE Chicken Serum 6% HSA 6% rHSA 0 20 40 60 80 100 120 0 20 40 60 80 100 120 DOC UREA TFE DOC UREA TFE DOC UREA TFE Chicken Serum 6% HSA 6% rHSA FSP VIF Absolute Tg Measure (fmol) tSIL IS cSIL IS SIL-rTg IS sTg cTg

There Are No Absolutes, Particularly in Protein QuantificationSurrogate matrices spiked with rTg served as external calibrators, while “unknown”samples were prepared by spiking

  • Upload
    others

  • View
    0

  • Download
    0

Embed Size (px)

Citation preview

Page 1: There Are No Absolutes, Particularly in Protein QuantificationSurrogate matrices spiked with rTg served as external calibrators, while “unknown”samples were prepared by spiking

There Are No Absolutes, Particularly in Protein Quantification 1. Center for Esoteric Testing, Laboratory Corporation of America® Holdings,

Burlington, North Carolina, 27215

2. MilliporeSigma, St. Louis, Missouri, 631033. MilliporeSigma, Round Rock, Texas, 78665

IntroductionQuantitative bottom-up proteomics has been termed “accurate” when using stable isotope-labeled (SIL)surrogates of the protein as a calibrator (i.e., internal calibration). These SIL materials are added as a peptidepre- or more conventionally post-digestion, as a cleavable (winged) peptide pre-digestion or as an intactproteoform prior to denaturation and digestion. This technique differs from classical externally calibrated,internally standardized (normalizing) assay workflows and forms the basis of this paper - is either techniqueaccurate? Fully-tryptic SIL peptides, cleavable SIL peptides, and a full-length SIL protein were compared asinternal calibrants and internal (normalizing) standards for quantifying 3 forms of unlabeled thyroglobulin (Tg) byprotein cleavage-isotope dilution mass spectrometry using multiple digestion conditions. All SIL materials andunlabeled proteins were standardized by amino acid analysis (NIST traceable) for assignment of accuracy.

MethodsFigure 1. Eight fully-tryptic SIL peptides (tSIL), 8 cleavable SIL peptides (cSIL), and SIL-rTg were compared asinternal calibrators and as internal (normalizing) standards for quantifying 3 samples of unlabeled Tg. The 3samples of unlabeled Tg quantified (in 0.1% human serum albumin, HSA) were BCR®457 (sTg, thyroglobulininternational reference material [1]), a commercially available purified human thyroglobulin (cTg), and a full-length recombinant protein (rTg) expressed and purified in identical fashion to the full-length SIL-rTg, but withoutlabeled amino acids. All SIL materials were labeled at different amino acid positions within the final signaturepeptide sequences to ensure selective LC-MS/MS analysis of each peptide-form in the same sample. Purity ofneat stock solutions of each SIL material and unlabeled Tg proteins were qualified by HPLC (peptides) or SDS-PAGE (proteins) and then standardized by triplicate amino acid analysis (AAA) using NIST amino acid calibrationmaterials (CV<5%). A NIST-traceable BSA control was run in parallel to qualify the accuracy of the AAA (Bias <2%,CV<5%).

Internal Calibration versus Internal Standardization

Absolute Digestion Efficiency

Uncontrolled Digestion-based Matrix Effects

References

Patricia L. Holland,1 Christopher M. Shuford,1 James J. Walters,2 Kevin Ray,2 Uma Sreenivasan,3 Sarah Aijaz,3 Russell P. Grant1

1. Feldt-Rasmussen, U. et al. Purification and Certification of Human Thyroglobulin Reference MaterialCRM457 Final Report; EUR 15611 EN; European Commission Community Bureau of Reference:Luxembourg, Brussels, 1994.

2. Konopka, A.; Boehm, M. E.; Rohmer, M.; Baeumlisberger, D.; Karas, M.; Lehmann, W. D. Anal. Bioanal.Chem. 2012, 404 (4), 1079–1087.

3. Heudi, O.; Barteau, S.; Zimmer, D.; Schmidt, J.; Bill, K.; Lehmann, N.; Bauer, C.; Kretz, O. Anal. Chem. 2008,80 (11), 4200–4207.

Figure 2. All working solutions were gravimetrically prepared to enable traceability to the original AAA-assignedvalues. For quantification, samples were prepared gravimetrically, then denatured and digested prior to LC-MS/MS analysis. SIL-rTg was added to samples prior to denaturation, cSIL peptides (purity corrected) prior todigestion, and tSIL peptides (purity corrected) after digestion. Additionally, 3 different denaturing conditions (plusDTT and heating) were tested including; 4 M urea (1M during digestion), 1% sodium deoxycholate (DOC, 0.25%during digestion), and 10% trifluoroethanol (TFE, 2.5% during digestion). For internal calibration, quantities of Tgwere calculated by taking the quotient of the unlabeled:SIL peptide peak areas measured and multiplying by theabsolute amount of the SIL calibrator added.

Conclusions

Recombinant versus Native Proteins

Peptide Internal Calibrators (Tryptic or Cleavable)• Do NOT facilitate absolute quantification due to < 100% digestion efficiency of native Tg• Do NOT facilitate inter-assay agreement due to variability in digestion efficiency (not commutable)• (Even small) impurities in cleavable peptide calibrators will confound standardization

Peptide Internal Standards (Tryptic or Cleavable)• Require external protein calibrators to achieve accuracy (matrix equivalency for calibration and sample)• Purity (concentration) is less important (requires no contribution to/from Native proteoforms)

Protein Internal Calibrators• Recombinant proteins are surrogate calibrators (at least the big ones)• Commutability not guaranteed (peptide, digestion, and IS dependent)• Only feasible method for accurate internal calibration (requires harmonization/response balancing of

recombinant SIL protein and may not agree with native protein)Protein Internal Standards

• Recombinant proteins are analogue internal standards (at least the big ones)• Only form of IS likely to normalize for digestion-based matrix effects (confounded by PTM’s and native

protein binding partners)• Only form of IS able to normalize for protein-enrichment variance (confounded by PTM’s and native

protein binding partners)

Figure 3. (a-c) Addition of SIL materials asinternal calibrators demonstrated bias > ±20%for the majority of peptides and denaturingconditions using tryptic peptides (tSIL).Cleavable (cSIL) internal calibrators yieldedincreased recoveries, with positive biasresulting from differential yield of labeledversus native peptide. Recombinant labeledprotein (SIL-rTg) as an internal calibratorprovided superior results, however, systematicbias versus all native forms was observed –even compared to recombinant native protein– supporting the hypothesis that labeledmaterials can digest differentially even whenexpressed identically (see rTg versus SIL-rTg[2]). (d-f) External calibration using unlabeledrTg with SIL materials added as internalstandards (I’sS) reduced measurement biasirrespective of the SIL IS added, albeit, certainpeptides (TFP) were not fully corrected.

Figure 4. Internal calibration using tSILs (Figure 3a) demonstrates that the resulting quantity isthe amount of peptide product produced via digestion rather than the starting amount ofprotein substrate. The digestion efficiency of the protein or cleavable substrate can thus bedetermined by comparing the amount of peptide product (Cpeptide, calculated using tSILcalibrators) to the absolute amount of substrate added (CSubstrate) to elucidate sources of bias.

𝐴𝑏𝑠𝑜𝑙𝑢𝑡𝑒 𝐷𝑖𝑔𝑒𝑠𝑡𝑖𝑜𝑛 𝐸𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 =𝐶𝑃𝑒𝑝𝑡𝑖𝑑𝑒

𝐶𝑆𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒× 100

The source of bias in Figure 3 is derived by the digestion efficiency differences between theunlabeled protein analytes and surrogate calibrants. Using the GGA peptide as an example, theGGA cSIL peptide demonstrates equivalent digestion efficiency versus native protein forms usingDOC and urea (Figure 4) – thus, resulting in no bias (Figure 3b); however, increased digestionefficiency was observed for the cSIL versus all 3 unlabeled proteins using trifluoroethanolresulting in positive bias in the accuracy determination (Figure 3b).

Figure 6. Disparity between the recombinant and human-derived Tg could arise throughdifferences in the PTM state of the 39 signature peptides rather than differences in digestionefficiency. Determination of the differences in glycosylation were evaluated (total glycancomposition, not positional glycosylation). Released glycans (a) MAN5; b) MAN6; c) G2F; d)MAN8; e) MAN9; f) G2FS; g) FA3S) from both the sTg (Left inset) and rTg (Right inset)demonstrate glycosylation of rTg with a modified profile (versus sTg), potentially impactingtertiary and quaternary structure together with digestion efficiency.

Figure 7. Digestion variance in potential calibrator matrices for human serum was studied. Surrogate matricesspiked with rTg served as external calibrators, while “unknown” samples were prepared by spiking 100 fmol sTgor cTg into Tg-deficient human serum. The three SIL materials (tSIL, cSIL, and SIL-rTg) were used as internalstandards for the FSP (top) and VIF (bottom) signature peptides. Results demonstrate using either human-derived HSA or recombinant HSA (rHSA) as surrogate matrices generated acceptable recoveries regardless of theinternal standard or digestion condition. A matrix effect was observed between human and chicken serum forthe FSP signature peptide (and not the VIF peptide) with DOC as the denaturant; resulting in negatively biasedquantification (≤80% recovery) of both sTg and cTg in human serum using the cSIL or tSIL ISs. Modification of thedenaturant conditions ameliorated this bias to some degree indicating the matrix effect is dependent on thecombination of digestion condition, signature peptide, and surrogate matrix selected.

tSIL Peptides cSIL PeptideSequence Purity Sequence Purity

FSPDDSAGASALLR 94.0% FYQRRRFSPDDSAGASALLRSGPYMP 78.1%

LEDIPVASLPDLHDIER 98.5% VTWKSRLEDIPVASLPDLHDIERALVGKD 59.7%

TFPAETIR 97.6% LHLDSKTFPAETIRFLQGDH 67.3%

VIFDANAPVAVR 97.0% KVPESKVIFDANAPVAVRSKVPDS 94.4%

VILEDK 100.0% ALFRKKVILEDKVKNFYT 92.0%

SQAIQVGTSWK 88.2% GRLLGRSQAIQVGTSWKQVDQFL 92.1%

GGADVASIHLLTAR 86.4% SLAADRGGADVASIHLLTARATNSQL 91.4%

EFSELLPNR 96.6% GGENYKEFSELLPNRQGLKKA 99.5%

[U-15

N, 13

C]-labeled residues

30 min, 56 °C10 mM DTT

+Denaturant

cleavable SIL (cSIL) peptide

unlabeld Tg

SIL-rTg

LC-SRM1.3 1.4 1.5 1.6 1.7

Retention Time (min)

fully-tryptic SIL (tSIL) peptide

tSILcSIL

3 pmol

Full-length (unlabeled Tg)

Full-length (SIL-rTg)

3 pmol

3 pmol (each)1.5 pmol(each)

0.1% HSA

30 min, 37 °C1:10 Substrate:EnzymeTPCK-treated Trypsin

0.0

1.0

2.0

3.0

4.0

5.0

6.0

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

FSP LED TFP VIF VIL SQA GGA EFS

sTg cTg rTg

0.0

1.0

2.0

3.0

4.0

5.0

6.0

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

FSP LED TFP VIF VIL SQA GGA EFS

0.0

1.0

2.0

3.0

4.0

5.0

6.0

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

FSP LED TFP VIF VIL SQA GGA EFS

Ab

solu

te T

g M

ea

sure

(p

mo

l)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

FSP LED TFP VIF VIL SQA GGA EFS

0.0

1.0

2.0

3.0

4.0

5.0

6.0

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

FSP LED TFP VIF VIL SQA GGA EFS

0.0

1.0

2.0

3.0

4.0

5.0

6.0

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

FSP LED TFP VIF VIL SQA GGA EFS

sTg cTg

Ab

solu

te T

g M

ea

sure

(p

mo

l)

a

b

c

d

e

f

FSP LED TFP VIF VIL SQA GGA EFS FSP LED TFP VIF VIL SQA GGA EFS

0%

20%

40%

60%

80%

100%

120%

DOC UREA TFE

Dig

estion E

ffic

iency

0%

20%

40%

60%

80%

100%

120%

DOC UREA TFE

Dig

estion E

ffic

iency

0%

20%

40%

60%

80%

100%

120%

DOC UREA TFE

Dig

estion E

ffic

iency

0%

20%

40%

60%

80%

100%

120%

DOC UREA TFE

Dig

estion E

ffic

iency

0%

20%

40%

60%

80%

100%

120%

DOC UREA TFE

Dig

estion E

ffic

iency

0%

20%

40%

60%

80%

100%

120%

DOC UREA TFE

Dig

estion E

ffic

iency

0%

20%

40%

60%

80%

100%

120%

DOC UREA TFE

Dig

estion E

ffic

iency

0%

20%

40%

60%

80%

100%

120%

DOC UREA TFE

Dig

estion E

ffic

iency

Ab

solu

te D

ige

stio

n E

ffic

ien

cy

EFSVIL SQA GGA

VIFFSP LED TFP

SIL-rTg cSILssTg cTg rTg

Figure 5. Additional quantification of BCR®457 (sTg) and catalog human-derived Tg (cTg) wasdetermined using 39 peptides with recombinant (rTg) as an external calibrator and SIL-rTg asthe internal standard. The reduced variance between peptides (CV = 5.7 – 7.5%) for rTg usingSIL-rTg is due to identical digestion efficiencies between unlabeled and SIL-rTg (i.e., commonrecombinant expression), akin to the analysis of biotherapeutics [3]. Increased variance wasobserved for the two human-derived Tg materials (sTg CV = 13.0 – 19.4%, cTg CV = 13.8 –17.9%), explained by disparity in the digestion efficiency of some signature peptides relative torTg and SIL-rTg, resulting in quantitative bias for those uncontrolled signature peptides.

0.50 0 1.50 0 2.50 0 3.50 0 4.50 0 5.50 0 6.50 0 7.50 0 8.50 0 9.50 0

0.00 0

1.00 0

2.00 0

3.00 0

4.00 0

5.00 0

6.00 0

0.0

1.0

2.0

3.0

4.0

5.0

6.0

sTg cTg rTg CS sTg cTg rTg CS sTg cTg rTg CS

DOC UREA TFE

Ab

solu

te T

g M

ea

sure

(p

mo

l)

sTg cTg rTg sTg cTg rTg sTg cTg rTg

Re

lati

ve In

ten

sity 100

80

60

40

20

sTg

ab c d

ef

g

15 25 35 45 5520 30 40 50 60

Retention Time (min)

rTg

a

b

cd

e

fg

15 25 35 45 5520 30 40 50 60

Retention Time (min)

0

0

20

40

60

80

100

120

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

ChickenSerum

6% HSA 6% rHSA

0

20

40

60

80

100

120

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

ChickenSerum

6% HSA 6% rHSA

0

20

40

60

80

100

120

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

ChickenSerum

6% HSA 6% rHSA

0

20

40

60

80

100

120

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

ChickenSerum

6% HSA 6% rHSA

0

20

40

60

80

100

120

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

ChickenSerum

6% HSA 6% rHSA

0

20

40

60

80

100

120

DO

C

UR

EA TFE

DO

C

UR

EA TFE

DO

C

UR

EA TFE

ChickenSerum

6% HSA 6% rHSA

FSP

VIF

Ab

solu

te T

g M

ea

sure

(fm

ol)

tSIL IS cSIL IS SIL-rTg IS

sTg cTg