Development of an All-Recombinant Intact Protein Standard for LC-MS Application Development and System Suitability Testing

No

. 64852

POSTER NOTE

RESULTS Preliminary R&D Mixture

The original protein mixture met the sample requirements of wide molecular weight range, widely distributed charge state envelopes across m/z, and representative diversity for quality control, application development, and method optimization. The R&D mixture, however, did not meet regulatory requirements with respect to animal origin components in the mixture, and no stability studies were done. In fact, degradation of certain proteins during storage was observed over time.

Figure 1. Workflow for recombinant protein mixture generation.

Figure 2. Original R&D mixture infusion-MS spectra. Top panel infusion spectra of R&D mixture at 140k resolution. Proteins from 12kD to 29kD are routinely detected. Bottom panel infusion spectral of R&D mixture at 8k resolution. All proteins typically detected, including the proteins at 42 and 66kD.

Table 1. Original R&D mixture composition, MW range, and m/z range

Figure 3. SDS-PAGE of several recombinant candidates for MS analysis.

1 Thioredoxin (Trx)2 - RNAseA3 - Protein G4 - Carboic Anhydrase5 - Protein AG6 Klennow

350 ng samples loaded to 12% SDS gel

OVERVIEW Purpose : develop a high quality, reproducible intact protein standard for LC-MS QC and application development.

Methods : expression and purification of recombinant proteins that meet MW, m/z distribution, purity, stability, reproducibility requirements of a sample used for LC and MS QC.

Results: a high quality, recombinant intact protein mixture for LC-MS quality control and application development was developed.

INTRODUCTION In recent years, interest in intact protein analysis by HPLC, LC-MS, and MSMS has increased significantly. This can be attributed to both improvements to LC and MS hardware, instrument control software, and data processing software. Conceptual shifts in how we can best address and answer biological questions given these emerging commercially available capabilities also explain this paradigm shift. Having witnessed the explosive growth of bottom-up proteomics and the subsequent evolution of high-quality, widely accessible standards to normalize platform performance in time and space, and assist with method development for new applications, we recognize a similar need for the Top-down proteomics field. Here, we describe the development of a high quality, recombinant, multi-purpose intact protein standard for LC, LC-MS, and LC-MSMS quality control and application development.

MATERIALS AND METHODSProtein standards were purchased from Sigma-Aldrich and evaluated by direct infusion ESI-MS on a Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap MS. Seven proteins were selected that 1) evenly covered a MW range of 12kD 66kD, 2) presented mostly clean, modification and adduct-free ESI spectra, and 3) whose ESI charge state distributions covered a wide m/z range from 500-2000. Mixing ratios were adjusted such that all seven proteins could be detected simultaneously in a single infusion MS experiment (R&D standard).A number of proteins candidates that were expected to mimic the characteristics of the proteins in the original R&D standard were then expressed in E. coli and B. subtills and purified. All were screened for ionization efficiency, purity, MW, charge state distribution by LC-MS, and infusion-MS, resulting in selection of the final candidates for the recombinant standard. Quality and stability of the selected proteins were verified by SDS-PAGE, UV HPLC, infusion-MS and LC-MS, and MSMS using a Q Exactive mass spectrometer. High resolution HCD spectra were collected to confirm the sequences assigned to the final protein list. From these confirmed sequences a standard FASTA file and flat file were created for distribution and easy analysis. Mixing ratios were again optimized and stability of the mixture was confirmed, again by SDS-PAGE, UV HPLC, infusion MS, and LC-MS. CONCLUSIONS -

A high quality intact protein standard was developed that meets needs for HPLC, MS, LC-MSMS quality control, method development, and optimization. The completely recombinant nature of the sample makes it compatible with clinical applications. The proteins cover a wide MW range, m/z range, and demonstrate good chromatographic separation.

ACKNOWLEDGEMENTS -We would like to acknowledge Jason Neil, Eugen Damoc, Johan Finell, Joanna Freeke, Suvi Ravella, and Ilja Ritamo for their help with early testing of, and feedback on, the R&D mixture.

We would like to thank the Kelleher lab for testing and feedback on candidate proteins for the final mixture.

TRADEMARKS/LICENSING - 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.

ProteinSigma

PN~MW(kD)

Center m/z

Cyochrome c C2506 12 773

RNAse A R6513 14 1369

Myoglobin M0630 17 808

Trypsin Inhibitor T6522 20 1110

Carbonic Anhydrase C2522 29 830

Enolase E6126 42 771

BSA A5611 66 1210

1. Transformation 2. Expression 3. Cell Lyses

6. Dialysis

7. Desalting

7 pr

otei

ns

9. Lyophilization

Protein Mix

5. 3 step Purification

8. 7 proteins

mix

4. Soluble proteins

Load Column

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z

700 800 900 1000 1100 1200 1300 1400 1500 1600 1700m/z

Figure 4. Final recombinant mixture infusion-MS spectra. Top panel infusion spectra of the optimized mixture using new recombinant protein candidates at 140k resolution. Trx, RNAseA, Protein G, and Carbonic Anhydrase are detected at this resolution setting. Bottom panel infusion spectral of new candidate mixture at 8k resolution. All proteins typically detected, including larger proteins, Protein AG and Klenow.

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700m/z

Development of an All-Recombinant Intact Protein Standard for LC-MS Application Development and System Suitability Testing

L

10 kDa

15 kDa

25 kDa

35 kDa

55 kDa

70 kDa

TrxRNAseA

rCA

ProtG

Klennow

ProtAG

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z

1 2 3 4 5 6

600 800 1000 1200 1400 1600 1800 2000m/z

851.5049z=14

600 800 1000 1200 1400 1600 1800 2000m/z

1244.8444z=11

600 800 1000 1200 1400 1600 1800 2000m/z

1016.6126z=21

600 700 800 900 1000 1100 1200 1300 1400 1500m/z

829.0857z=35

600 800 1000 1200 1400 1600 1800 2000m/z

1030.81

600 700 800 900 1000 1100 1200 1300 1400 1500m/z

756.59

400 600 800 1000 1200 1400m/z

400 600 800 1000 1200 1400 1600 1800 2000m/z

300 500 700 900 1100 1300 1500m/z

400 600 800 1000 1200 1400m/z

300 500 700 900 1100 1300 1500m/z

Figure 3. MS analysis of protein candidates. Left panel shows the full scan spectrum of each candidate protein collected on a Thermo Scientific Q Exactive HF MS with an inset showing deconvolution results from Thermo Scientific Protein Deconvolution 3.0 software. In some cases, oxidation or sodiation are evident. Work is ongoing to reduce sodiation. The right hand panel shows HCD spectra of each protein collected on a Q Exactive HF along with Thermo Scientific ProSightPC 3.0 software results (inset). The ProSight results from the largest protein, Klennow, were derived from a high resolution CID spectra from the Thermo Scientific Orbitrap Fusion Lumos Tribrid MS.

NL: 4.44E6

600 800 1000 1200 1400 1600m/z

NL: 4.51E6

600 800 1000 1200 1400 1600m/z

Fresh 7 days @ 37C Figure 7. Preliminary

stability studies. Shown here is Protein G, reconsituted fresh or after 7 days incubated at 37C, corresponding to 1 year storage at -20C.

400 600 800 1000 1200 1400 1600m/z

Trx

RNAseA

Protein G

Carbonic Anhydrase

Protein AG

Klenow

Helene L. Cardasis1, Rosa Viner2, Vikrant Gohil1, Kay Opperman3, Shane Bechler4, John Rogers3, Kelly Flook4, Egle Capkauske5, Kestutis Bargaila5, Agne Alminaite5, Viktorija Vitkovske5, Paul Haney31 - Thermo Fisher Scientific, Cambridge, MA 02139; 2- Thermo Fisher Scientific, San Jose, CA 95134; 3- Thermo Fisher Scientific, Rockford, IL 61101; 4- Thermo Fisher Scientific, Sunnyvale, CA 94085; 5- Thermo Fisher Scientific, Vilnius, Lithuania LT-02241

3.00 3.75 5.00 6.25 7.50 8.75 10.00 11.25 13.00

-10.0

0.0

12.5

25.0

37.5

50.0

62.5

75.0

87.5

100.0

min

mAU

Protein G

TRX

KlenowProtein AG

Carbonic Anhydrase

Protein kDa

TRX 12

ProteinG 21.4

Carbonic Anhydrase

29.1

Protein AG 50.1

Klenow 68.1

Figure 5. UV-HPLC analysis of recombinant proteins. MAbPac RP 2.1 x 50mm column; mobile phase A: Water + 0.1% Formic acid, B: 80/20 (v/v) ACN/ Water + 0.1% Formic acid; Flow Rate: 0.3 mL/min; 5-80% B in 15 minutes; Temperature: 80C; Sample: 0.2mg/mL protein (see chromatogram); Injection: 10 L; Detection: UV 280 nm.

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0-4.00

-2.50

-1.25

0.00

1.25

2.50

3.75

5.00

6.25

7.50

8.00

Time, minutes

mAU

TRX

Protein G

KlenowProtein

AG

Carbonic Anhydrase

Protein kDa

TRX 12

ProteinG 21.4

Carbonic Anhydrase 29.1

Protein AG 50.1

Klenow 68.1

Figure 6. UV-HPLC analysis of recombinant proteins. Thermo Scientific UltiMate 3000 RSLCnano System; Column: Thermo Scientific ProSwift RP-4H LC 200m x 25cm; Mobile Phase A: Water + 0.1% Formic acid, B: 80/20 (v/v) ACN/ Water + 0.1% Formic acid; Flow Rate: 5 L/min; 5-80% B in 15 minutes; Temperature: 30C; Sample: 0.2mg/mL protein (see chromatogram); Injection: 0.1 L. Detection: UV 214 nm

RESULTS New Recombinant Mixture

RESULTS Preliminary R&D Mixture

The original protein mixture met the sample requirements of wide molecular weight range, widely distributed charge state envelopes across m/z, and representative diversity for quality control, application development, and method optimization. The R&D mixture, however, did not meet regulatory requirements with respect to animal origin components in the mixture, and no stability studies were done. In fact, degradation of certain proteins during storage was observed over time.

Figure 1. Workflow for recombinant protein mixture generation.

Figure 2. Original R&D mixture infusion-MS spectra. Top panel infusion spectra of R&D mixture at 140k resolution. Proteins from 12kD to 29kD are routinely detected. Bottom panel infusion spectral of R&D mixture at 8k resolution. All proteins typically detected, including the proteins at 42 and 66kD.

Table 1. Original R&D mixture composition, MW range, and m/z range

Figure 3. SDS-PAGE of several recombinant candidates for MS analysis.

1 Thioredoxin (Trx)2 - RNAseA3 - Protein G4 - Carboic Anhydrase5 - Protein AG6 Klennow

350 ng samples loaded to 12% SDS gel

OVERVIEW Purpose : develop a high quality, reproducible intact protein standard for LC-MS QC and application development.

Methods : expression and purification of recombinant proteins that meet MW, m/z distribution, purity, stability, reproducibility requirements of a sample used for LC and MS QC.

Results: a high quality, recombinant intact protein mixture for LC-MS quality control and application development was developed.

INTRODUCTION In recent years, interest in intact protein analysis by HPLC, LC-MS, and MSMS has increased significantly. This can be attributed to both improvements to LC and MS hardware, instrument control software, and data processing software. Conceptual shifts in how we can best address and answer biological questions given these emerging commercially available capabilities also explain this paradigm shift. Having witnessed the explosive growth of bottom-up proteomics and the subsequent evolution of high-quality, widely accessible standards to normalize platform performance in time and space, and assist with method development for new applications, we recognize a similar need for the Top-down proteomics field. Here, we describe the development of a high quality, recombinant, multi-purpose intact protein standard for LC, LC-MS, and LC-MSMS quality control and application development.

MATERIALS AND METHODSProtein standards were purchased from Sigma-Aldrich and evaluated by direct infusion ESI-MS on a Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap MS. Seven proteins were selected that 1) evenly covered a MW range of 12kD 66kD, 2) presented mostly clean, modification and adduct-free ESI spectra, and 3) whose ESI charge state distributions covered a wide m/z range from 500-2000. Mixing ratios were adjusted such that all seven proteins could be detected simultaneously in a single infusion MS experiment (R&D standard).A number of proteins candidates that were expected to mimic the characteristics of the proteins in the original R&D standard were then expressed in E. coli and B. subtills and purified. All were screened for ionization efficiency, purity, MW, charge state distribution by LC-MS, and infusion-MS, resulting in selection of the final candidates for the recombinant standard. Quality and stability of the selected proteins were verified by SDS-PAGE, UV HPLC, infusion-MS and LC-MS, and MSMS using a Q Exactive mass spectrometer. High resolution HCD spectra were collected to confirm the sequences assigned to the final protein list. From these confirmed sequences a standard FASTA file and flat file were created for distribution and easy analysis. Mixing ratios were again optimized and stability of the mixture was confirmed, again by SDS-PAGE, UV HPLC, infusion MS, and LC-MS. CONCLUSIONS -

A high quality intact protein standard was developed that meets needs for HPLC, MS, LC-MSMS quality control, method development, and optimization. The completely recombinant nature of the sample makes it compatible with clinical applications. The proteins cover a wide MW range, m/z range, and demonstrate good chromatographic separation.

ACKNOWLEDGEMENTS -We would like to acknowledge Jason Neil, Eugen Damoc, Johan Finell, Joanna Freeke, Suvi Ravella, and Ilja Ritamo for their help with early testing of, and feedback on, the R&D mixture.

We would like to thank the Kelleher lab for testing and feedback on candidate proteins for the final mixture.

TRADEMARKS/LICENSING - 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.

ProteinSigma

PN~MW(kD)

Center m/z

Cyochrome c C2506 12 773

RNAse A R6513 14 1369

Myoglobin M0630 17 808

Trypsin Inhibitor T6522 20 1110

Carbonic Anhydrase C2522 29 830

Enolase E6126 42 771

BSA A5611 66 1210

1. Transformation 2. Expression 3. Cell Lyses

6. Dialysis

7. Desalting

7 pr

otei

ns

9. Lyophilization

Protein Mix

5. 3 step Purification

8. 7 proteins

mix

4. Soluble proteins

Load Column

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z

700 800 900 1000 1100 1200 1300 1400 1500 1600 1700m/z

Figure 4. Final recombinant mixture infusion-MS spectra. Top panel infusion spectra of the optimized mixture using new recombinant protein candidates at 140k resolution. Trx, RNAseA, Protein G, and Carbonic Anhydrase are detected at this resolution setting. Bottom panel infusion spectral of new candidate mixture at 8k resolution. All proteins typically detected, including larger proteins, Protein AG and Klenow.

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700m/z

Development of an All-Recombinant Intact Protein Standard for LC-MS Application Development and System Suitability Testing

L

10 kDa

15 kDa

25 kDa

35 kDa

55 kDa

70 kDa

TrxRNAseA

rCA

ProtG

Klennow

ProtAG

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z

1 2 3 4 5 6

600 800 1000 1200 1400 1600 1800 2000m/z

851.5049z=14

600 800 1000 1200 1400 1600 1800 2000m/z

1244.8444z=11

600 800 1000 1200 1400 1600 1800 2000m/z

1016.6126z=21

600 700 800 900 1000 1100 1200 1300 1400 1500m/z

829.0857z=35

600 800 1000 1200 1400 1600 1800 2000m/z

1030.81

600 700 800 900 1000 1100 1200 1300 1400 1500m/z

756.59

400 600 800 1000 1200 1400m/z

400 600 800 1000 1200 1400 1600 1800 2000m/z

300 500 700 900 1100 1300 1500m/z

400 600 800 1000 1200 1400m/z

300 500 700 900 1100 1300 1500m/z

Figure 3. MS analysis of protein candidates. Left panel shows the full scan spectrum of each candidate protein collected on a Thermo Scientific Q Exactive HF MS with an inset showing deconvolution results from Thermo Scientific Protein Deconvolution 3.0 software. In some cases, oxidation or sodiation are evident. Work is ongoing to reduce sodiation. The right hand panel shows HCD spectra of each protein collected on a Q Exactive HF along with Thermo Scientific ProSightPC 3.0 software results (inset). The ProSight results from the largest protein, Klennow, were derived from a high resolution CID spectra from the Thermo Scientific Orbitrap Fusion Lumos Tribrid MS.

NL: 4.44E6

600 800 1000 1200 1400 1600m/z

NL: 4.51E6

600 800 1000 1200 1400 1600m/z

Fresh 7 days @ 37C Figure 7. Preliminary

stability studies. Shown here is Protein G, reconsituted fresh or after 7 days incubated at 37C, corresponding to 1 year storage at -20C.

400 600 800 1000 1200 1400 1600m/z

Trx

RNAseA

Protein G

Carbonic Anhydrase

Protein AG

Klenow

Helene L. Cardasis1, Rosa Viner2, Vikrant Gohil1, Kay Opperman3, Shane Bechler4, John Rogers3, Kelly Flook4, Egle Capkauske5, Kestutis Bargaila5, Agne Alminaite5, Viktorija Vitkovske5, Paul Haney31 - Thermo Fisher Scientific, Cambridge, MA 02139; 2- Thermo Fisher Scientific, San Jose, CA 95134; 3- Thermo Fisher Scientific, Rockford, IL 61101; 4- Thermo Fisher Scientific, Sunnyvale, CA 94085; 5- Thermo Fisher Scientific, Vilnius, Lithuania LT-02241

3.00 3.75 5.00 6.25 7.50 8.75 10.00 11.25 13.00

-10.0

0.0

12.5

25.0

37.5

50.0

62.5

75.0

87.5

100.0

min

mAU

Protein G

TRX

KlenowProtein AG

Carbonic Anhydrase

Protein kDa

TRX 12

ProteinG 21.4

Carbonic Anhydrase

29.1

Protein AG 50.1

Klenow 68.1

Figure 5. UV-HPLC analysis of recombinant proteins. MAbPac RP 2.1 x 50mm column; mobile phase A: Water + 0.1% Formic acid, B: 80/20 (v/v) ACN/ Water + 0.1% Formic acid; Flow Rate: 0.3 mL/min; 5-80% B in 15 minutes; Temperature: 80C; Sample: 0.2mg/mL protein (see chromatogram); Injection: 10 L; Detection: UV 280 nm.

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0-4.00

-2.50

-1.25

0.00

1.25

2.50

3.75

5.00

6.25

7.50

8.00

Time, minutes

mAU

TRX

Protein G

KlenowProtein

AG

Carbonic Anhydrase

Protein kDa

TRX 12

ProteinG 21.4

Carbonic Anhydrase 29.1

Protein AG 50.1

Klenow 68.1

Figure 6. UV-HPLC analysis of recombinant proteins. Thermo Scientific UltiMate 3000 RSLCnano System; Column: Thermo Scientific ProSwift RP-4H LC 200m x 25cm; Mobile Phase A: Water + 0.1% Formic acid, B: 80/20 (v/v) ACN/ Water + 0.1% Formic acid; Flow Rate: 5 L/min; 5-80% B in 15 minutes; Temperature: 30C; Sample: 0.2mg/mL protein (see chromatogram); Injection: 0.1 L. Detection: UV 214 nm

RESULTS New Recombinant Mixture

RESULTS Preliminary R&D Mixture

The original protein mixture met the sample requirements of wide molecular weight range, widely distributed charge state envelopes across m/z, and representative diversity for quality control, application development, and method optimization. The R&D mixture, however, did not meet regulatory requirements with respect to animal origin components in the mixture, and no stability studies were done. In fact, degradation of certain proteins during storage was observed over time.

Figure 1. Workflow for recombinant protein mixture generation.

Figure 2. Original R&D mixture infusion-MS spectra. Top panel infusion spectra of R&D mixture at 140k resolution. Proteins from 12kD to 29kD are routinely detected. Bottom panel infusion spectral of R&D mixture at 8k resolution. All proteins typically detected, including the proteins at 42 and 66kD.

Table 1. Original R&D mixture composition, MW range, and m/z range

Figure 3. SDS-PAGE of several recombinant candidates for MS analysis.

1 Thioredoxin (Trx)2 - RNAseA3 - Protein G4 - Carboic Anhydrase5 - Protein AG6 Klennow

350 ng samples loaded to 12% SDS gel

OVERVIEW Purpose : develop a high quality, reproducible intact protein standard for LC-MS QC and application development.

Methods : expression and purification of recombinant proteins that meet MW, m/z distribution, purity, stability, reproducibility requirements of a sample used for LC and MS QC.

Results: a high quality, recombinant intact protein mixture for LC-MS quality control and application development was developed.

INTRODUCTION In recent years, interest in intact protein analysis by HPLC, LC-MS, and MSMS has increased significantly. This can be attributed to both improvements to LC and MS hardware, instrument control software, and data processing software. Conceptual shifts in how we can best address and answer biological questions given these emerging commercially available capabilities also explain this paradigm shift. Having witnessed the explosive growth of bottom-up proteomics and the subsequent evolution of high-quality, widely accessible standards to normalize platform performance in time and space, and assist with method development for new applications, we recognize a similar need for the Top-down proteomics field. Here, we describe the development of a high quality, recombinant, multi-purpose intact protein standard for LC, LC-MS, and LC-MSMS quality control and application development.

MATERIALS AND METHODSProtein standards were purchased from Sigma-Aldrich and evaluated by direct infusion ESI-MS on a Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap MS. Seven proteins were selected that 1) evenly covered a MW range of 12kD 66kD, 2) presented mostly clean, modification and adduct-free ESI spectra, and 3) whose ESI charge state distributions covered a wide m/z range from 500-2000. Mixing ratios were adjusted such that all seven proteins could be detected simultaneously in a single infusion MS experiment (R&D standard).A number of proteins candidates that were expected to mimic the characteristics of the proteins in the original R&D standard were then expressed in E. coli and B. subtills and purified. All were screened for ionization efficiency, purity, MW, charge state distribution by LC-MS, and infusion-MS, resulting in selection of the final candidates for the recombinant standard. Quality and stability of the selected proteins were verified by SDS-PAGE, UV HPLC, infusion-MS and LC-MS, and MSMS using a Q Exactive mass spectrometer. High resolution HCD spectra were collected to confirm the sequences assigned to the final protein list. From these confirmed sequences a standard FASTA file and flat file were created for distribution and easy analysis. Mixing ratios were again optimized and stability of the mixture was confirmed, again by SDS-PAGE, UV HPLC, infusion MS, and LC-MS. CONCLUSIONS -

A high quality intact protein standard was developed that meets needs for HPLC, MS, LC-MSMS quality control, method development, and optimization. The completely recombinant nature of the sample makes it compatible with clinical applications. The proteins cover a wide MW range, m/z range, and demonstrate good chromatographic separation.

ACKNOWLEDGEMENTS -We would like to acknowledge Jason Neil, Eugen Damoc, Johan Finell, Joanna Freeke, Suvi Ravella, and Ilja Ritamo for their help with early testing of, and feedback on, the R&D mixture.

We would like to thank the Kelleher lab for testing and feedback on candidate proteins for the final mixture.

TRADEMARKS/LICENSING - 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.

ProteinSigma

PN~MW(kD)

Center m/z

Cyochrome c C2506 12 773

RNAse A R6513 14 1369

Myoglobin M0630 17 808

Trypsin Inhibitor T6522 20 1110

Carbonic Anhydrase C2522 29 830

Enolase E6126 42 771

BSA A5611 66 1210

1. Transformation 2. Expression 3. Cell Lyses

6. Dialysis

7. Desalting

7 pr

otei

ns

9. Lyophilization

Protein Mix

5. 3 step Purification

8. 7 proteins

mix

4. Soluble proteins

Load Column

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z

700 800 900 1000 1100 1200 1300 1400 1500 1600 1700m/z

Figure 4. Final recombinant mixture infusion-MS spectra. Top panel infusion spectra of the optimized mixture using new recombinant protein candidates at 140k resolution. Trx, RNAseA, Protein G, and Carbonic Anhydrase are detected at this resolution setting. Bottom panel infusion spectral of new candidate mixture at 8k resolution. All proteins typically detected, including larger proteins, Protein AG and Klenow.

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700m/z

Development of an All-Recombinant Intact Protein Standard for LC-MS Application Development and System Suitability Testing

L

10 kDa

15 kDa

25 kDa

35 kDa

55 kDa

70 kDa

TrxRNAseA

rCA

ProtG

Klennow

ProtAG

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z

1 2 3 4 5 6

600 800 1000 1200 1400 1600 1800 2000m/z

851.5049z=14

600 800 1000 1200 1400 1600 1800 2000m/z

1244.8444z=11

600 800 1000 1200 1400 1600 1800 2000m/z

1016.6126z=21

600 700 800 900 1000 1100 1200 1300 1400 1500m/z

829.0857z=35

600 800 1000 1200 1400 1600 1800 2000m/z

1030.81

600 700 800 900 1000 1100 1200 1300 1400 1500m/z

756.59

400 600 800 1000 1200 1400m/z

400 600 800 1000 1200 1400 1600 1800 2000m/z

300 500 700 900 1100 1300 1500m/z

400 600 800 1000 1200 1400m/z

300 500 700 900 1100 1300 1500m/z

Figure 3. MS analysis of protein candidates. Left panel shows the full scan spectrum of each candidate protein collected on a Thermo Scientific Q Exactive HF MS with an inset showing deconvolution results from Thermo Scientific Protein Deconvolution 3.0 software. In some cases, oxidation or sodiation are evident. Work is ongoing to reduce sodiation. The right hand panel shows HCD spectra of each protein collected on a Q Exactive HF along with Thermo Scientific ProSightPC 3.0 software results (inset). The ProSight results from the largest protein, Klennow, were derived from a high resolution CID spectra from the Thermo Scientific Orbitrap Fusion Lumos Tribrid MS.

NL: 4.44E6

600 800 1000 1200 1400 1600m/z

NL: 4.51E6

600 800 1000 1200 1400 1600m/z

Fresh 7 days @ 37C Figure 7. Preliminary

stability studies. Shown here is Protein G, reconsituted fresh or after 7 days incubated at 37C, corresponding to 1 year storage at -20C.

400 600 800 1000 1200 1400 1600m/z

Trx

RNAseA

Protein G

Carbonic Anhydrase

Protein AG

Klenow

Helene L. Cardasis1, Rosa Viner2, Vikrant Gohil1, Kay Opperman3, Shane Bechler4, John Rogers3, Kelly Flook4, Egle Capkauske5, Kestutis Bargaila5, Agne Alminaite5, Viktorija Vitkovske5, Paul Haney31 - Thermo Fisher Scientific, Cambridge, MA 02139; 2- Thermo Fisher Scientific, San Jose, CA 95134; 3- Thermo Fisher Scientific, Rockford, IL 61101; 4- Thermo Fisher Scientific, Sunnyvale, CA 94085; 5- Thermo Fisher Scientific, Vilnius, Lithuania LT-02241

3.00 3.75 5.00 6.25 7.50 8.75 10.00 11.25 13.00

-10.0

0.0

12.5

25.0

37.5

50.0

62.5

75.0

87.5

100.0

min

mAU

Protein G

TRX

KlenowProtein AG

Carbonic Anhydrase

Protein kDa

TRX 12

ProteinG 21.4

Carbonic Anhydrase

29.1

Protein AG 50.1

Klenow 68.1

Figure 5. UV-HPLC analysis of recombinant proteins. MAbPac RP 2.1 x 50mm column; mobile phase A: Water + 0.1% Formic acid, B: 80/20 (v/v) ACN/ Water + 0.1% Formic acid; Flow Rate: 0.3 mL/min; 5-80% B in 15 minutes; Temperature: 80C; Sample: 0.2mg/mL protein (see chromatogram); Injection: 10 L; Detection: UV 280 nm.

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0-4.00

-2.50

-1.25

0.00

1.25

2.50

3.75

5.00

6.25

7.50

8.00

Time, minutes

mAU

TRX

Protein G

KlenowProtein

AG

Carbonic Anhydrase

Protein kDa

TRX 12

ProteinG 21.4

Carbonic Anhydrase 29.1

Protein AG 50.1

Klenow 68.1

Figure 6. UV-HPLC analysis of recombinant proteins. Thermo Scientific UltiMate 3000 RSLCnano System; Column: Thermo Scientific ProSwift RP-4H LC 200m x 25cm; Mobile Phase A: Water + 0.1% Formic acid, B: 80/20 (v/v) ACN/ Water + 0.1% Formic acid; Flow Rate: 5 L/min; 5-80% B in 15 minutes; Temperature: 30C; Sample: 0.2mg/mL protein (see chromatogram); Injection: 0.1 L. Detection: UV 214 nm

RESULTS New Recombinant Mixture

Helene L. Cardasis1, Rosa Viner2, Vikrant Gohil1, Kay Opperman3, Shane Bechler4, John Rogers3, Kelly Flook4, Egle Capkauske5, Kestutis Bargaila5, Agne Alminaite5, Viktorija Vitkovske5, Paul Haney31Thermo Fisher Scienti c, Cambridge, MA; 2Thermo Fisher Scienti c, San Jose, CA; 3Thermo Fisher Scienti c, Rockford, IL; 4Thermo Fisher Scienti c, Sunnyvale, CA; 5Thermo Fisher Scienti c, Vilnius, Lithuania

RESULTS Preliminary R&D Mixture

The original protein mixture met the sample requirements of wide molecular weight range, widely distributed charge state envelopes across m/z, and representative diversity for quality control, application development, and method optimization. The R&D mixture, however, did not meet regulatory requirements with respect to animal origin components in the mixture, and no stability studies were done. In fact, degradation of certain proteins during storage was observed over time.

Figure 1. Workflow for recombinant protein mixture generation.

Figure 2. Original R&D mixture infusion-MS spectra. Top panel infusion spectra of R&D mixture at 140k resolution. Proteins from 12kD to 29kD are routinely detected. Bottom panel infusion spectral of R&D mixture at 8k resolution. All proteins typically detected, including the proteins at 42 and 66kD.

Table 1. Original R&D mixture composition, MW range, and m/z range

Figure 3. SDS-PAGE of several recombinant candidates for MS analysis.

1 Thioredoxin (Trx)2 - RNAseA3 - Protein G4 - Carboic Anhydrase5 - Protein AG6 Klennow

350 ng samples loaded to 12% SDS gel

OVERVIEW Purpose : develop a high quality, reproducible intact protein standard for LC-MS QC and application development.

Methods : expression and purification of recombinant proteins that meet MW, m/z distribution, purity, stability, reproducibility requirements of a sample used for LC and MS QC.

Results: a high quality, recombinant intact protein mixture for LC-MS quality control and application development was developed.

INTRODUCTION In recent years, interest in intact protein analysis by HPLC, LC-MS, and MSMS has increased significantly. This can be attributed to both improvements to LC and MS hardware, instrument control software, and data processing software. Conceptual shifts in how we can best address and answer biological questions given these emerging commercially available capabilities also explain this paradigm shift. Having witnessed the explosive growth of bottom-up proteomics and the subsequent evolution of high-quality, widely accessible standards to normalize platform performance in time and space, and assist with method development for new applications, we recognize a similar need for the Top-down proteomics field. Here, we describe the development of a high quality, recombinant, multi-purpose intact protein standard for LC, LC-MS, and LC-MSMS quality control and application development.

MATERIALS AND METHODSProtein standards were purchased from Sigma-Aldrich and evaluated by direct infusion ESI-MS on a Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap MS. Seven proteins were selected that 1) evenly covered a MW range of 12kD 66kD, 2) presented mostly clean, modification and adduct-free ESI spectra, and 3) whose ESI charge state distributions covered a wide m/z range from 500-2000. Mixing ratios were adjusted such that all seven proteins could be detected simultaneously in a single infusion MS experiment (R&D standard).A number of proteins candidates that were expected to mimic the characteristics of the proteins in the original R&D standard were then expressed in E. coli and B. subtills and purified. All were screened for ionization efficiency, purity, MW, charge state distribution by LC-MS, and infusion-MS, resulting in selection of the final candidates for the recombinant standard. Quality and stability of the selected proteins were verified by SDS-PAGE, UV HPLC, infusion-MS and LC-MS, and MSMS using a Q Exactive mass spectrometer. High resolution HCD spectra were collected to confirm the sequences assigned to the final protein list. From these confirmed sequences a standard FASTA file and flat file were created for distribution and easy analysis. Mixing ratios were again optimized and stability of the mixture was confirmed, again by SDS-PAGE, UV HPLC, infusion MS, and LC-MS. CONCLUSIONS -

A high quality intact protein standard was developed that meets needs for HPLC, MS, LC-MSMS quality control, method development, and optimization. The completely recombinant nature of the sample makes it compatible with clinical applications. The proteins cover a wide MW range, m/z range, and demonstrate good chromatographic separation.

ACKNOWLEDGEMENTS -We would like to acknowledge Jason Neil, Eugen Damoc, Johan Finell, Joanna Freeke, Suvi Ravella, and Ilja Ritamo for their help with early testing of, and feedback on, the R&D mixture.

We would like to thank the Kelleher lab for testing and feedback on candidate proteins for the final mixture.

TRADEMARKS/LICENSING - 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.

ProteinSigma

PN~MW(kD)

Center m/z

Cyochrome c C2506 12 773

RNAse A R6513 14 1369

Myoglobin M0630 17 808

Trypsin Inhibitor T6522 20 1110

Carbonic Anhydrase C2522 29 830

Enolase E6126 42 771

BSA A5611 66 1210

1. Transformation 2. Expression 3. Cell Lyses

6. Dialysis

7. Desalting

7 pr

otei

ns

9. Lyophilization

Protein Mix

5. 3 step Purification

8. 7 proteins

mix

4. Soluble proteins

Load Column

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z

700 800 900 1000 1100 1200 1300 1400 1500 1600 1700m/z

Figure 4. Final recombinant mixture infusion-MS spectra. Top panel infusion spectra of the optimized mixture using new recombinant protein candidates at 140k resolution. Trx, RNAseA, Protein G, and Carbonic Anhydrase are detected at this resolution setting. Bottom panel infusion spectral of new candidate mixture at 8k resolution. All proteins typically detected, including larger proteins, Protein AG and Klenow.

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700m/z

Development of an All-Recombinant Intact Protein Standard for LC-MS Application Development and System Suitability Testing

L

10 kDa

15 kDa

25 kDa

35 kDa

55 kDa

70 kDa

TrxRNAseA

rCA

ProtG

Klennow

ProtAG

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z

1 2 3 4 5 6

600 800 1000 1200 1400 1600 1800 2000m/z

851.5049z=14

600 800 1000 1200 1400 1600 1800 2000m/z

1244.8444z=11

600 800 1000 1200 1400 1600 1800 2000m/z

1016.6126z=21

600 700 800 900 1000 1100 1200 1300 1400 1500m/z

829.0857z=35

600 800 1000 1200 1400 1600 1800 2000m/z

1030.81

600 700 800 900 1000 1100 1200 1300 1400 1500m/z

756.59

400 600 800 1000 1200 1400m/z

400 600 800 1000 1200 1400 1600 1800 2000m/z

300 500 700 900 1100 1300 1500m/z

400 600 800 1000 1200 1400m/z

300 500 700 900 1100 1300 1500m/z

Figure 3. MS analysis of protein candidates. Left panel shows the full scan spectrum of each candidate protein collected on a Thermo Scientific Q Exactive HF MS with an inset showing deconvolution results from Thermo Scientific Protein Deconvolution 3.0 software. In some cases, oxidation or sodiation are evident. Work is ongoing to reduce sodiation. The right hand panel shows HCD spectra of each protein collected on a Q Exactive HF along with Thermo Scientific ProSightPC 3.0 software results (inset). The ProSight results from the largest protein, Klennow, were derived from a high resolution CID spectra from the Thermo Scientific Orbitrap Fusion Lumos Tribrid MS.

NL: 4.44E6

600 800 1000 1200 1400 1600m/z

NL: 4.51E6

600 800 1000 1200 1400 1600m/z

Fresh 7 days @ 37C Figure 7. Preliminary

stability studies. Shown here is Protein G, reconsituted fresh or after 7 days incubated at 37C, corresponding to 1 year storage at -20C.

400 600 800 1000 1200 1400 1600m/z

Trx

RNAseA

Protein G

Carbonic Anhydrase

Protein AG

Klenow

Helene L. Cardasis1, Rosa Viner2, Vikrant Gohil1, Kay Opperman3, Shane Bechler4, John Rogers3, Kelly Flook4, Egle Capkauske5, Kestutis Bargaila5, Agne Alminaite5, Viktorija Vitkovske5, Paul Haney31 - Thermo Fisher Scientific, Cambridge, MA 02139; 2- Thermo Fisher Scientific, San Jose, CA 95134; 3- Thermo Fisher Scientific, Rockford, IL 61101; 4- Thermo Fisher Scientific, Sunnyvale, CA 94085; 5- Thermo Fisher Scientific, Vilnius, Lithuania LT-02241

3.00 3.75 5.00 6.25 7.50 8.75 10.00 11.25 13.00

-10.0

0.0

12.5

25.0

37.5

50.0

62.5

75.0

87.5

100.0

min

mAU

Protein G

TRX

KlenowProtein AG

Carbonic Anhydrase

Protein kDa

TRX 12

ProteinG 21.4

Carbonic Anhydrase

29.1

Protein AG 50.1

Klenow 68.1

Figure 5. UV-HPLC analysis of recombinant proteins. MAbPac RP 2.1 x 50mm column; mobile phase A: Water + 0.1% Formic acid, B: 80/20 (v/v) ACN/ Water + 0.1% Formic acid; Flow Rate: 0.3 mL/min; 5-80% B in 15 minutes; Temperature: 80C; Sample: 0.2mg/mL protein (see chromatogram); Injection: 10 L; Detection: UV 280 nm.

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0-4.00

-2.50

-1.25

0.00

1.25

2.50

3.75

5.00

6.25

7.50

8.00

Time, minutes

mAU

TRX

Protein G

KlenowProtein

AG

Carbonic Anhydrase

Protein kDa

TRX 12

ProteinG 21.4

Carbonic Anhydrase 29.1

Protein AG 50.1

Klenow 68.1

Figure 6. UV-HPLC analysis of recombinant proteins. Thermo Scientific UltiMate 3000 RSLCnano System; Column: Thermo Scientific ProSwift RP-4H LC 200m x 25cm; Mobile Phase A: Water + 0.1% Formic acid, B: 80/20 (v/v) ACN/ Water + 0.1% Formic acid; Flow Rate: 5 L/min; 5-80% B in 15 minutes; Temperature: 30C; Sample: 0.2mg/mL protein (see chromatogram); Injection: 0.1 L. Detection: UV 214 nm

RESULTS New Recombinant Mixture

RESULTS Preliminary R&D Mixture

The original protein mixture met the sample requirements of wide molecular weight range, widely distributed charge state envelopes across m/z, and representative diversity for quality control, application development, and method optimization. The R&D mixture, however, did not meet regulatory requirements with respect to animal origin components in the mixture, and no stability studies were done. In fact, degradation of certain proteins during storage was observed over time.

Figure 1. Workflow for recombinant protein mixture generation.

Figure 2. Original R&D mixture infusion-MS spectra. Top panel infusion spectra of R&D mixture at 140k resolution. Proteins from 12kD to 29kD are routinely detected. Bottom panel infusion spectral of R&D mixture at 8k resolution. All proteins typically detected, including the proteins at 42 and 66kD.

Table 1. Original R&D mixture composition, MW range, and m/z range

Figure 3. SDS-PAGE of several recombinant candidates for MS analysis.

1 Thioredoxin (Trx)2 - RNAseA3 - Protein G4 - Carboic Anhydrase5 - Protein AG6 Klennow

350 ng samples loaded to 12% SDS gel

OVERVIEW Purpose : develop a high quality, reproducible intact protein standard for LC-MS QC and application development.

Methods : expression and purification of recombinant proteins that meet MW, m/z distribution, purity, stability, reproducibility requirements of a sample used for LC and MS QC.

Results: a high quality, recombinant intact protein mixture for LC-MS quality control and application development was developed.

INTRODUCTION In recent years, interest in intact protein analysis by HPLC, LC-MS, and MSMS has increased significantly. This can be attributed to both improvements to LC and MS hardware, instrument control software, and data processing software. Conceptual shifts in how we can best address and answer biological questions given these emerging commercially available capabilities also explain this paradigm shift. Having witnessed the explosive growth of bottom-up proteomics and the subsequent evolution of high-quality, widely accessible standards to normalize platform performance in time and space, and assist with method development for new applications, we recognize a similar need for the Top-down proteomics field. Here, we describe the development of a high quality, recombinant, multi-purpose intact protein standard for LC, LC-MS, and LC-MSMS quality control and application development.

MATERIALS AND METHODSProtein standards were purchased from Sigma-Aldrich and evaluated by direct infusion ESI-MS on a Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap MS. Seven proteins were selected that 1) evenly covered a MW range of 12kD 66kD, 2) presented mostly clean, modification and adduct-free ESI spectra, and 3) whose ESI charge state distributions covered a wide m/z range from 500-2000. Mixing ratios were adjusted such that all seven proteins could be detected simultaneously in a single infusion MS experiment (R&D standard).A number of proteins candidates that were expected to mimic the characteristics of the proteins in the original R&D standard were then expressed in E. coli and B. subtills and purified. All were screened for ionization efficiency, purity, MW, charge state distribution by LC-MS, and infusion-MS, resulting in selection of the final candidates for the recombinant standard. Quality and stability of the selected proteins were verified by SDS-PAGE, UV HPLC, infusion-MS and LC-MS, and MSMS using a Q Exactive mass spectrometer. High resolution HCD spectra were collected to confirm the sequences assigned to the final protein list. From these confirmed sequences a standard FASTA file and flat file were created for distribution and easy analysis. Mixing ratios were again optimized and stability of the mixture was confirmed, again by SDS-PAGE, UV HPLC, infusion MS, and LC-MS. CONCLUSIONS -

A high quality intact protein standard was developed that meets needs for HPLC, MS, LC-MSMS quality control, method development, and optimization. The completely recombinant nature of the sample makes it compatible with clinical applications. The proteins cover a wide MW range, m/z range, and demonstrate good chromatographic separation.

ACKNOWLEDGEMENTS -We would like to acknowledge Jason Neil, Eugen Damoc, Johan Finell, Joanna Freeke, Suvi Ravella, and Ilja Ritamo for their help with early testing of, and feedback on, the R&D mixture.

We would like to thank the Kelleher lab for testing and feedback on candidate proteins for the final mixture.

TRADEMARKS/LICENSING - 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.

ProteinSigma

PN~MW(kD)

Center m/z

Cyochrome c C2506 12 773

RNAse A R6513 14 1369

Myoglobin M0630 17 808

Trypsin Inhibitor T6522 20 1110

Carbonic Anhydrase C2522 29 830

Enolase E6126 42 771

BSA A5611 66 1210

1. Transformation 2. Expression 3. Cell Lyses

6. Dialysis

7. Desalting

7 pr

otei

ns

9. Lyophilization

Protein Mix

5. 3 step Purification

8. 7 proteins

mix

4. Soluble proteins

Load Column

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z

700 800 900 1000 1100 1200 1300 1400 1500 1600 1700m/z

Figure 4. Final recombinant mixture infusion-MS spectra. Top panel infusion spectra of the optimized mixture using new recombinant protein candidates at 140k resolution. Trx, RNAseA, Protein G, and Carbonic Anhydrase are detected at this resolution setting. Bottom panel infusion spectral of new candidate mixture at 8k resolution. All proteins typically detected, including larger proteins, Protein AG and Klenow.

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700m/z

Development of an All-Recombinant Intact Protein Standard for LC-MS Application Development and System Suitability Testing

L

10 kDa

15 kDa

25 kDa

35 kDa

55 kDa

70 kDa

TrxRNAseA

rCA

ProtG

Klennow

ProtAG

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z

1 2 3 4 5 6

600 800 1000 1200 1400 1600 1800 2000m/z

851.5049z=14

600 800 1000 1200 1400 1600 1800 2000m/z

1244.8444z=11

600 800 1000 1200 1400 1600 1800 2000m/z

1016.6126z=21

600 700 800 900 1000 1100 1200 1300 1400 1500m/z

829.0857z=35

600 800 1000 1200 1400 1600 1800 2000m/z

1030.81

600 700 800 900 1000 1100 1200 1300 1400 1500m/z

756.59

400 600 800 1000 1200 1400m/z

400 600 800 1000 1200 1400 1600 1800 2000m/z

300 500 700 900 1100 1300 1500m/z

400 600 800 1000 1200 1400m/z

300 500 700 900 1100 1300 1500m/z

Figure 3. MS analysis of protein candidates. Left panel shows the full scan spectrum of each candidate protein collected on a Thermo Scientific Q Exactive HF MS with an inset showing deconvolution results from Thermo Scientific Protein Deconvolution 3.0 software. In some cases, oxidation or sodiation are evident. Work is ongoing to reduce sodiation. The right hand panel shows HCD spectra of each protein collected on a Q Exactive HF along with Thermo Scientific ProSightPC 3.0 software results (inset). The ProSight results from the largest protein, Klennow, were derived from a high resolution CID spectra from the Thermo Scientific Orbitrap Fusion Lumos Tribrid MS.

NL: 4.44E6

600 800 1000 1200 1400 1600m/z

NL: 4.51E6

600 800 1000 1200 1400 1600m/z

Fresh 7 days @ 37C Figure 7. Preliminary

stability studies. Shown here is Protein G, reconsituted fresh or after 7 days incubated at 37C, corresponding to 1 year storage at -20C.

400 600 800 1000 1200 1400 1600m/z

Trx

RNAseA

Protein G

Carbonic Anhydrase

Protein AG

Klenow

Helene L. Cardasis1, Rosa Viner2, Vikrant Gohil1, Kay Opperman3, Shane Bechler4, John Rogers3, Kelly Flook4, Egle Capkauske5, Kestutis Bargaila5, Agne Alminaite5, Viktorija Vitkovske5, Paul Haney31 - Thermo Fisher Scientific, Cambridge, MA 02139; 2- Thermo Fisher Scientific, San Jose, CA 95134; 3- Thermo Fisher Scientific, Rockford, IL 61101; 4- Thermo Fisher Scientific, Sunnyvale, CA 94085; 5- Thermo Fisher Scientific, Vilnius, Lithuania LT-02241

3.00 3.75 5.00 6.25 7.50 8.75 10.00 11.25 13.00

-10.0

0.0

12.5

25.0

37.5

50.0

62.5

75.0

87.5

100.0

min

mAU

Protein G

TRX

KlenowProtein AG

Carbonic Anhydrase

Protein kDa

TRX 12

ProteinG 21.4

Carbonic Anhydrase

29.1

Protein AG 50.1

Klenow 68.1

Figure 5. UV-HPLC analysis of recombinant proteins. MAbPac RP 2.1 x 50mm column; mobile phase A: Water + 0.1% Formic acid, B: 80/20 (v/v) ACN/ Water + 0.1% Formic acid; Flow Rate: 0.3 mL/min; 5-80% B in 15 minutes; Temperature: 80C; Sample: 0.2mg/mL protein (see chromatogram); Injection: 10 L; Detection: UV 280 nm.

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0-4.00

-2.50

-1.25

0.00

1.25

2.50

3.75

5.00

6.25

7.50

8.00

Time, minutes

mAU

TRX

Protein G

KlenowProtein

AG

Carbonic Anhydrase

Protein kDa

TRX 12

ProteinG 21.4

Carbonic Anhydrase 29.1

Protein AG 50.1

Klenow 68.1

Figure 6. UV-HPLC analysis of recombinant proteins. Thermo Scientific UltiMate 3000 RSLCnano System; Column: Thermo Scientific ProSwift RP-4H LC 200m x 25cm; Mobile Phase A: Water + 0.1% Formic acid, B: 80/20 (v/v) ACN/ Water + 0.1% Formic acid; Flow Rate: 5 L/min; 5-80% B in 15 minutes; Temperature: 30C; Sample: 0.2mg/mL protein (see chromatogram); Injection: 0.1 L. Detection: UV 214 nm

RESULTS New Recombinant Mixture

Find out more at thermo sher.com 2016 Thermo Fisher Scienti c Inc. All rights reserved. Lipid Search is a registered trademark of MKI. All trademarks are the property of Thermo Fisher Scienti c and its subsidiaries unless otherwise speci ed. PN64852-EN 08/16S

For Research Use Only. Not to be used in diagnostic procedures.

RESULTS Preliminary R&D Mixture

The original protein mixture met the sample requirements of wide molecular weight range, widely distributed charge state envelopes across m/z, and representative diversity for quality control, application development, and method optimization. The R&D mixture, however, did not meet regulatory requirements with respect to animal origin components in the mixture, and no stability studies were done. In fact, degradation of certain proteins during storage was observed over time.

Figure 1. Workflow for recombinant protein mixture generation.

Figure 2. Original R&D mixture infusion-MS spectra. Top panel infusion spectra of R&D mixture at 140k resolution. Proteins from 12kD to 29kD are routinely detected. Bottom panel infusion spectral of R&D mixture at 8k resolution. All proteins typically detected, including the proteins at 42 and 66kD.

Table 1. Original R&D mixture composition, MW range, and m/z range

Figure 3. SDS-PAGE of several recombinant candidates for MS analysis.

1 Thioredoxin (Trx)2 - RNAseA3 - Protein G4 - Carboic Anhydrase5 - Protein AG6 Klennow

350 ng samples loaded to 12% SDS gel

OVERVIEW Purpose : develop a high quality, reproducible intact protein standard for LC-MS QC and application development.

Methods : expression and purification of recombinant proteins that meet MW, m/z distribution, purity, stability, reproducibility requirements of a sample used for LC and MS QC.

Results: a high quality, recombinant intact protein mixture for LC-MS quality control and application development was developed.

INTRODUCTION In recent years, interest in intact protein analysis by HPLC, LC-MS, and MSMS has increased significantly. This can be attributed to both improvements to LC and MS hardware, instrument control software, and data processing software. Conceptual shifts in how we can best address and answer biological questions given these emerging commercially available capabilities also explain this paradigm shift. Having witnessed the explosive growth of bottom-up proteomics and the subsequent evolution of high-quality, widely accessible standards to normalize platform performance in time and space, and assist with method development for new applications, we recognize a similar need for the Top-down proteomics field. Here, we describe the development of a high quality, recombinant, multi-purpose intact protein standard for LC, LC-MS, and LC-MSMS quality control and application development.

MATERIALS AND METHODSProtein standards were purchased from Sigma-Aldrich and evaluated by direct infusion ESI-MS on a Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap MS. Seven proteins were selected that 1) evenly covered a MW range of 12kD 66kD, 2) presented mostly clean, modification and adduct-free ESI spectra, and 3) whose ESI charge state distributions covered a wide m/z range from 500-2000. Mixing ratios were adjusted such that all seven proteins could be detected simultaneously in a single infusion MS experiment (R&D standard).A number of proteins candidates that were expected to mimic the characteristics of the proteins in the original R&D standard were then expressed in E. coli and B. subtills and purified. All were screened for ionization efficiency, purity, MW, charge state distribution by LC-MS, and infusion-MS, resulting in selection of the final candidates for the recombinant standard. Quality and stability of the selected proteins were verified by SDS-PAGE, UV HPLC, infusion-MS and LC-MS, and MSMS using a Q Exactive mass spectrometer. High resolution HCD spectra were collected to confirm the sequences assigned to the final protein list. From these confirmed sequences a standard FASTA file and flat file were created for distribution and easy analysis. Mixing ratios were again optimized and stability of the mixture was confirmed, again by SDS-PAGE, UV HPLC, infusion MS, and LC-MS. CONCLUSIONS -

A high quality intact protein standard was developed that meets needs for HPLC, MS, LC-MSMS quality control, method development, and optimization. The completely recombinant nature of the sample makes it compatible with clinical applications. The proteins cover a wide MW range, m/z range, and demonstrate good chromatographic separation.

ACKNOWLEDGEMENTS -We would like to acknowledge Jason Neil, Eugen Damoc, Johan Finell, Joanna Freeke, Suvi Ravella, and Ilja Ritamo for their help with early testing of, and feedback on, the R&D mixture.

We would like to thank the Kelleher lab for testing and feedback on candidate proteins for the final mixture.

TRADEMARKS/LICENSING - 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.

ProteinSigma

PN~MW(kD)

Center m/z

Cyochrome c C2506 12 773

RNAse A R6513 14 1369

Myoglobin M0630 17 808

Trypsin Inhibitor T6522 20 1110

Carbonic Anhydrase C2522 29 830

Enolase E6126 42 771

BSA A5611 66 1210

1. Transformation 2. Expression 3. Cell Lyses

6. Dialysis

7. Desalting

7 pr

otei

ns

9. Lyophilization

Protein Mix

5. 3 step Purification

8. 7 proteins

mix

4. Soluble proteins

Load Column

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z

700 800 900 1000 1100 1200 1300 1400 1500 1600 1700m/z

Figure 4. Final recombinant mixture infusion-MS spectra. Top panel infusion spectra of the optimized mixture using new recombinant protein candidates at 140k resolution. Trx, RNAseA, Protein G, and Carbonic Anhydrase are detected at this resolution setting. Bottom panel infusion spectral of new candidate mixture at 8k resolution. All proteins typically detected, including larger proteins, Protein AG and Klenow.

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700m/z

Development of an All-Recombinant Intact Protein Standard for LC-MS Application Development and System Suitability Testing

L

10 kDa

15 kDa

25 kDa

35 kDa

55 kDa

70 kDa

TrxRNAseA

rCA

ProtG

Klennow

ProtAG

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z

1 2 3 4 5 6

600 800 1000 1200 1400 1600 1800 2000m/z

851.5049z=14

600 800 1000 1200 1400 1600 1800 2000m/z

1244.8444z=11

600 800 1000 1200 1400 1600 1800 2000m/z

1016.6126z=21

600 700 800 900 1000 1100 1200 1300 1400 1500m/z

829.0857z=35

600 800 1000 1200 1400 1600 1800 2000m/z

1030.81

600 700 800 900 1000 1100 1200 1300 1400 1500m/z

756.59

400 600 800 1000 1200 1400m/z

400 600 800 1000 1200 1400 1600 1800 2000m/z

300 500 700 900 1100 1300 1500m/z

400 600 800 1000 1200 1400m/z

300 500 700 900 1100 1300 1500m/z

Figure 3. MS analysis of protein candidates. Left panel shows the full scan spectrum of each candidate protein collected on a Thermo Scientific Q Exactive HF MS with an inset showing deconvolution results from Thermo Scientific Protein Deconvolution 3.0 software. In some cases, oxidation or sodiation are evident. Work is ongoing to reduce sodiation. The right hand panel shows HCD spectra of each protein collected on a Q Exactive HF along with Thermo Scientific ProSightPC 3.0 software results (inset). The ProSight results from the largest protein, Klennow, were derived from a high resolution CID spectra from the Thermo Scientific Orbitrap Fusion Lumos Tribrid MS.

NL: 4.44E6

600 800 1000 1200 1400 1600m/z

NL: 4.51E6

600 800 1000 1200 1400 1600m/z

Fresh 7 days @ 37C Figure 7. Preliminary

stability studies. Shown here is Protein G, reconsituted fresh or after 7 days incubated at 37C, corresponding to 1 year storage at -20C.

400 600 800 1000 1200 1400 1600m/z

Trx

RNAseA

Protein G

Carbonic Anhydrase

Protein AG

Klenow

Helene L. Cardasis1, Rosa Viner2, Vikrant Gohil1, Kay Opperman3, Shane Bechler4, John Rogers3, Kelly Flook4, Egle Capkauske5, Kestutis Bargaila5, Agne Alminaite5, Viktorija Vitkovske5, Paul Haney31 - Thermo Fisher Scientific, Cambridge, MA 02139; 2- Thermo Fisher Scientific, San Jose, CA 95134; 3- Thermo Fisher Scientific, Rockford, IL 61101; 4- Thermo Fisher Scientific, Sunnyvale, CA 94085; 5- Thermo Fisher Scientific, Vilnius, Lithuania LT-02241

3.00 3.75 5.00 6.25 7.50 8.75 10.00 11.25 13.00

-10.0

0.0

12.5

25.0

37.5

50.0

62.5

75.0

87.5

100.0

min

mAU

Protein G

TRX

KlenowProtein AG

Carbonic Anhydrase

Protein kDa

TRX 12

ProteinG 21.4

Carbonic Anhydrase

29.1

Protein AG 50.1

Klenow 68.1

Figure 5. UV-HPLC analysis of recombinant proteins. MAbPac RP 2.1 x 50mm column; mobile phase A: Water + 0.1% Formic acid, B: 80/20 (v/v) ACN/ Water + 0.1% Formic acid; Flow Rate: 0.3 mL/min; 5-80% B in 15 minutes; Temperature: 80C; Sample: 0.2mg/mL protein (see chromatogram); Injection: 10 L; Detection: UV 280 nm.

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0-4.00

-2.50

-1.25

0.00

1.25

2.50

3.75

5.00

6.25

7.50

8.00

Time, minutes

mAU

TRX

Protein G

KlenowProtein

AG

Carbonic Anhydrase

Protein kDa

TRX 12

ProteinG 21.4

Carbonic Anhydrase 29.1

Protein AG 50.1

Klenow 68.1

Figure 6. UV-HPLC analysis of recombinant proteins. Thermo Scientific UltiMate 3000 RSLCnano System; Column: Thermo Scientific ProSwift RP-4H LC 200m x 25cm; Mobile Phase A: Water + 0.1% Formic acid, B: 80/20 (v/v) ACN/ Water + 0.1% Formic acid; Flow Rate: 5 L/min; 5-80% B in 15 minutes; Temperature: 30C; Sample: 0.2mg/mL protein (see chromatogram); Injection: 0.1 L. Detection: UV 214 nm

RESULTS New Recombinant Mixture

RESULTS Preliminary R&D Mixture

The original protein mixture met the sample requirements of wide molecular weight range, widely distributed charge state envelopes across m/z, and representative diversity for quality control, application development, and method optimization. The R&D mixture, however, did not meet regulatory requirements with respect to animal origin components in the mixture, and no stability studies were done. In fact, degradation of certain proteins during storage was observed over time.

Figure 1. Workflow for recombinant protein mixture generation.

Figure 2. Original R&D mixture infusion-MS spectra. Top panel infusion spectra of R&D mixture at 140k resolution. Proteins from 12kD to 29kD are routinely detected. Bottom panel infusion spectral of R&D mixture at 8k resolution. All proteins typically detected, including the proteins at 42 and 66kD.

Table 1. Original R&D mixture composition, MW range, and m/z range

Figure 3. SDS-PAGE of several recombinant candidates for MS analysis.

1 Thioredoxin (Trx)2 - RNAseA3 - Protein G4 - Carboic Anhydrase5 - Protein AG6 Klennow

350 ng samples loaded to 12% SDS gel

OVERVIEW Purpose : develop a high quality, reproducible intact protein standard for LC-MS QC and application development.

Methods : expression and purification of recombinant proteins that meet MW, m/z distribution, purity, stability, reproducibility requirements of a sample used for LC and MS QC.

Results: a high quality, recombinant intact protein mixture for LC-MS quality control and application development was developed.

INTRODUCTION In recent years, interest in intact protein analysis by HPLC, LC-MS, and MSMS has increased significantly. This can be attributed to both improvements to LC and MS hardware, instrument control software, and data processing software. Conceptual shifts in how we can best address and answer biological questions given these emerging commercially available capabilities also explain this paradigm shift. Having witnessed the explosive growth of bottom-up proteomics and the subsequent evolution of high-quality, widely accessible standards to normalize platform performance in time and space, and assist with method development for new applications, we recognize a similar need for the Top-down proteomics field. Here, we describe the development of a high quality, recombinant, multi-purpose intact protein standard for LC, LC-MS, and LC-MSMS quality control and application development.

MATERIALS AND METHODSProtein standards were purchased from Sigma-Aldrich and evaluated by direct infusion ESI-MS on a Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap MS. Seven proteins were selected that 1) evenly covered a MW range of 12kD 66kD, 2) presented mostly clean, modification and adduct-free ESI spectra, and 3) whose ESI charge state distributions covered a wide m/z range from 500-2000. Mixing ratios were adjusted such that all seven proteins could be detected simultaneously in a single infusion MS experiment (R&D standard).A number of proteins candidates that were expected to mimic the characteristics of the proteins in the original R&D standard were then expressed in E. coli and B. subtills and purified. All were screened for ionization efficiency, purity, MW, charge state distribution by LC-MS, and infusion-MS, resulting in selection of the final candidates for the recombinant standard. Quality and stability of the selected proteins were verified by SDS-PAGE, UV HPLC, infusion-MS and LC-MS, and MSMS using a Q Exactive mass spectrometer. High resolution HCD spectra were collected to confirm the sequences assigned to the final protein list. From these confirmed sequences a standard FASTA file and flat file were created for distribution and easy analysis. Mixing ratios were again optimized and stability of the mixture was confirmed, again by SDS-PAGE, UV HPLC, infusion MS, and LC-MS. CONCLUSIONS -

A high quality intact protein standard was developed that meets needs for HPLC, MS, LC-MSMS quality control, method development, and optimization. The completely recombinant nature of the sample makes it compatible with clinical applications. The proteins cover a wide MW range, m/z range, and demonstrate good chromatographic separation.

ACKNOWLEDGEMENTS -We would like to acknowledge Jason Neil, Eugen Damoc, Johan Finell, Joanna Freeke, Suvi Ravella, and Ilja Ritamo for their help with early testing of, and feedback on, the R&D mixture.

We would like to thank the Kelleher lab for testing and feedback on candidate proteins for the final mixture.

TRADEMARKS/LICENSING - 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.

ProteinSigma

PN~MW(kD)

Center m/z

Cyochrome c C2506 12 773

RNAse A R6513 14 1369

Myoglobin M0630 17 808

Trypsin Inhibitor T6522 20 1110

Carbonic Anhydrase C2522 29 830

Enolase E6126 42 771

BSA A5611 66 1210

1. Transformation 2. Expression 3. Cell Lyses

6. Dialysis

7. Desalting

7 pr

otei

ns

9. Lyophilization

Protein Mix

5. 3 step Purification

8. 7 proteins

mix

4. Soluble proteins

Load Column

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z

700 800 900 1000 1100 1200 1300 1400 1500 1600 1700m/z

Figure 4. Final recombinant mixture infusion-MS spectra. Top panel infusion spectra of the optimized mixture using new recombinant protein candidates at 140k resolution. Trx, RNAseA, Protein G, and Carbonic Anhydrase are detected at this resolution setting. Bottom panel infusion spectral of new candidate mixture at 8k resolution. All proteins typically detected, including larger proteins, Protein AG and Klenow.

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700m/z

Development of an All-Recombinant Intact Protein Standard for LC-MS Application Development and System Suitability Testing

L

10 kDa

15 kDa

25 kDa

35 kDa

55 kDa

70 kDa

TrxRNAseA

rCA

ProtG

Klennow

ProtAG

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z

1 2 3 4 5 6

600 800 1000 1200 1400 1600 1800 2000m/z

851.5049z=14

600 800 1000 1200 1400 1600 1800 2000m/z

1244.8444z=11

600 800 1000 1200 1400 1600 1800 2000m/z

1016.6126z=21

600 700 800 900 1000 1100 1200 1300 1400 1500m/z

829.0857z=35

600 800 1000 1200 1400 1600 1800 2000m/z

1030.81

600 700 800 900 1000 1100 1200 1300 1400 1500m/z

756.59

400 600 800 1000 1200 1400m/z

400 600 800 1000 1200 1400 1600 1800 2000m/z

300 500 700 900 1100 1300 1500m/z

400 600 800 1000 1200 1400m/z

300 500 700 900 1100 1300 1500m/z

Figure 3. MS analysis of protein candidates. Left panel shows the full scan spectrum of each candidate protein collected on a Thermo Scientific Q Exactive HF MS with an inset showing deconvolution results from Thermo Scientific Protein Deconvolution 3.0 software. In some cases, oxidation or sodiation are evident. Work is ongoing to reduce sodiation. The right hand panel shows HCD spectra of each protein collected on a Q Exactive HF along with Thermo Scientific ProSightPC 3.0 software results (inset). The ProSight results from the largest protein, Klennow, were derived from a high resolution CID spectra from the Thermo Scientific Orbitrap Fusion Lumos Tribrid MS.

NL: 4.44E6

600 800 1000 1200 1400 1600m/z

NL: 4.51E6

600 800 1000 1200 1400 1600m/z

Fresh 7 days @ 37C Figure 7. Preliminary

stability studies. Shown here is Protein G, reconsituted fresh or after 7 days incubated at 37C, corresponding to 1 year storage at -20C.

400 600 800 1000 1200 1400 1600m/z

Trx

RNAseA

Protein G

Carbonic Anhydrase

Protein AG

Klenow

Helene L. Cardasis1, Rosa Viner2, Vikrant Gohil1, Kay Opperman3, Shane Bechler4, John Rogers3, Kelly Flook4, Egle Capkauske5, Kestutis Bargaila5, Agne Alminaite5, Viktorija Vitkovske5, Paul Haney31 - Thermo Fisher Scientific, Cambridge, MA 02139; 2- Thermo Fisher Scientific, San Jose, CA 95134; 3- Thermo Fisher Scientific, Rockford, IL 61101; 4- Thermo Fisher Scientific, Sunnyvale, CA 94085; 5- Thermo Fisher Scientific, Vilnius, Lithuania LT-02241

3.00 3.75 5.00 6.25 7.50 8.75 10.00 11.25 13.00

-10.0

0.0

12.5

25.0

37.5

50.0

62.5

75.0

87.5

100.0

min

mAU

Protein G

TRX

KlenowProtein AG

Carbonic Anhydrase

Protein kDa

TRX 12

ProteinG 21.4

Carbonic Anhydrase

29.1

Protein AG 50.1

Klenow 68.1

Figure 5. UV-HPLC analysis of recombinant proteins. MAbPac RP 2.1 x 50mm column; mobile phase A: Water + 0.1% Formic acid, B: 80/20 (v/v) ACN/ Water + 0.1% Formic acid; Flow Rate: 0.3 mL/min; 5-80% B in 15 minutes; Temperature: 80C; Sample: 0.2mg/mL protein (see chromatogram); Injection: 10 L; Detection: UV 280 nm.

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0-4.00

-2.50

-1.25

0.00

1.25

2.50

3.75

5.00

6.25

7.50

8.00

Time, minutes

mAU

TRX

Protein G

KlenowProtein

AG

Carbonic Anhydrase

Protein kDa

TRX 12

ProteinG 21.4

Carbonic Anhydrase 29.1

Protein AG 50.1

Klenow 68.1

Figure 6. UV-HPLC analysis of recombinant proteins. Thermo Scientific UltiMate 3000 RSLCnano System; Column: Thermo Scientific ProSwift RP-4H LC 200m x 25cm; Mobile Phase A: Water + 0.1% Formic acid, B: 80/20 (v/v) ACN/ Water + 0.1% Formic acid; Flow Rate: 5 L/min; 5-80% B in 15 minutes; Temperature: 30C; Sample: 0.2mg/mL protein (see chromatogram); Injection: 0.1 L. Detection: UV 214 nm

RESULTS New Recombinant Mixture