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Peptide Bioanalysis
Shiva Kumar Gudlawar
Senior Analyst
Buhoot Research Lab
Dubai
1
Overview
• Introduction
• Sample handling
• Why LCMS?
• Approach Using LCMS
• Case study
2
Synonyms
• Large molecules
• Macromolecules
• Biotherapeutics
• Biopharamceuticals
• Biologics
3
Biotherapeutics
• Monoclonal antibodies
• Therapeutic peptides
• Antibody drug conjugates(ADCs)
• Carrier conjugates
• Mes...
Introduction
• Importance of Bio therapeutics drugs have increased enormously during
recent years owing to their high spec...
Sample handling
6
Solubility
• General rules in predicting Solubility
• Peptides shorter than 5 amino acids are generally aqueous soluble
• ...
Stability
• Plasma contains preoteases which degrades protein or peptide by acting at amide
linkage.
• DEP(Diisopropylfluo...
Non Specific Binding
• Peptides often exhibit adsorption to vials and tubes at low
concentration.
• Responsible for incons...
Why LCMS?
10
Major Techniques (LCMS, LBA)
• Advantages of LCMS over LBA
• Selectivity between similar peptides and proteins (quality in...
12
LCMS-Approach for peptide bioanalysis
13
Quantification through intact protein analysis.
• This approach is the true measurement of whole protein.
• MS uses SRM by...
Surrogate peptide approach
• Commonly employed method for quantification of proteins.
• Usually signature peptides which a...
Protein digestion:
• Different enzymes used for digestion (trypsin, Glu-C, Lys-C, chymotrypsin, Asp-N)
• Trypsin is common...
Sample clean up.
• Plasma contains more than 60% of Albumin and 30—35% of immunoiglobulins.
• Achieving analyte separation...
Sample clean Up Cont…
• Generally larger protein precipitate around 40% of organic and smaller peptides
may need higher % ...
Chromatography
• For most LC seperations traditional C18 columns are good enough
• Recent advances in column technology of...
Mass spectrometry.
• Peptide and proteins generally multiply charged in ES source
• Ratio of formation one charge state to...
Challenges using LCMS
• Multiple charging and wide isotopic distribution restricts sensitivity
• Flow rate and solvent wil...
Method validation
22
Method Validation cont….
• For LBS 4-6-20 rule applies(4 out of 6 should be within 20% of nominal value)
• For LCMS 4-6-15...
Case Study
Quantification of Peptide (Compound_T) in
Plasma by HRMS
(Orbitrap Fusion_Ultima 3000)
24
Blast to look for signature peptide
• https://www.expasy.org/proteomics
• http://www.uniprot.org/
25
Signature peptide (Insilico digest)
26
Signature peptide (Blast)
27
Native form of the Peptide
201216_IP_01 #37 RT: 0.13 AV: 1 SB: 946 0.65-3.95 NL: 2.83E6
F: FTMS + p ESI Full ms [197.0777-...
Reduced using DTT (600c for 30 min)
291216_TRIAL_IP_PEP_013 #1438 RT: 6.00 AV: 1 NL: 8.62E6
F: FTMS + p ESI Full ms [197.0...
Reduced and alkylated using Iodoacetamide
201216_BLK_05 #640 RT: 2.12 AV: 1 NL: 8.87E7
F: FTMS + p ESI Full ms [197.0777-1...
Trypsin digest_peptide map(confirmation of
sequence)
RT: 0.00 - 35.00
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Tim...
MSMS spectra for peptide(DCLGWFK)-
463.2154(M+H)2+
080117_ext_TRIAL_041 #1886 RT: 3.95 AV: 1 SB: 245 3.37-3.85 , 4.05-5.09...
MSMS spectra for peptide(YKLW)-
305.1734(M+H)2+
080117_ext_TRIAL_041 #1858 RT: 3.89 AV: 1 SB: 244 3.37-3.85 , 4.05-5.09 NL...
Blank and LLOQ (1 ng/ml) of two unique peptides
RT: 0.00 - 8.00
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7....
Chromatograms of different concentrations for
peptide YKLW
RT: 0.00 - 8.00
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5...
Points to consider
• pH to be maintained at around 8 for Activity of DTT and Trypsin
• Percentage of organic modifier can ...
Conclusion
• Quantification using HRMS for proteins and large molecules gives better
selectivity and sensitivity.
• HRMS g...
References
• http://www.ionsource.com/
• https://www.expasy.org/proteomics
• Therapeutic peptide bioanalysis_Sciex applica...
Acknowledgements
• Mrs. Huda Zaheer
• Mrs. Amrita Rajagopal
• Mr. Aazam Syed
• Dr. Dhanapal swaminathan
39
Large Meets Small
Thank You
40
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Peptide_Bioanalysis (1)

  1. 1. Peptide Bioanalysis Shiva Kumar Gudlawar Senior Analyst Buhoot Research Lab Dubai 1
  2. 2. Overview • Introduction • Sample handling • Why LCMS? • Approach Using LCMS • Case study 2
  3. 3. Synonyms • Large molecules • Macromolecules • Biotherapeutics • Biopharamceuticals • Biologics 3
  4. 4. Biotherapeutics • Monoclonal antibodies • Therapeutic peptides • Antibody drug conjugates(ADCs) • Carrier conjugates • Messenger RNA therapeutics • Fusion proteins 4
  5. 5. Introduction • Importance of Bio therapeutics drugs have increased enormously during recent years owing to their high specificity and low toxicity. • They vary from small peptides to large proteins to fusion proteins. • Market valued at around US$199 billion in 2013 projected to grow by 13.5% by 2020 • More than 600 biological drugs are currently approved and now account for 30% of all drugs in development. • For Bioanalysis we segregate peptides from proteins based on size. Molecular weight above 6000 Da classified as proteins. • Many of therapeutic drugs are below 6000 Da so classified as peptides. 5
  6. 6. Sample handling 6
  7. 7. Solubility • General rules in predicting Solubility • Peptides shorter than 5 amino acids are generally aqueous soluble • Peptides containing <25% of hydrophobic amino acids are aqueous soluble. • Hydrophobic peptides containing 50% or more of hydrophobic amino acids are insoluble or partially aqueous soluble. These peptides needs to be dissolved in DMSO, DMF or acetonitrile before making aqueous dilutions. • Peptides containing >75% of hydrophobic amino acids (DEHKNQRSTY) capable of forming gels these peptides are solubilized in organic solvents or pH of the buffer to be adjusted. 7
  8. 8. Stability • Plasma contains preoteases which degrades protein or peptide by acting at amide linkage. • DEP(Diisopropylfluorophosphate), Cocktails (Sodium fluoride, potassium oxalate, Trichloro acetic acid), pefabloc (better than DEP) • Choice of stabilizer is compound dependant. • Even acidifying with formic acid or TFA will inhibit proteases. • Storage conditions like -20, -800C needs to be evaluated • Use ice baths where ever possible to slow down the reaction. • Plasma to be separated within half hour of blood collection and stored in conditions specified. 8
  9. 9. Non Specific Binding • Peptides often exhibit adsorption to vials and tubes at low concentration. • Responsible for inconsistent results. • Use of low adsoption polypropylene tubes, silica deactivated glass are suggested for making dilutions. • To minimize the adsorption serial dilutions can be prepared in plasma • For matrix free solutions solubility may be enhanced by aqueous- organic mixtures, adding BSA or surfactants to block adsorption sites • For extracted samples organic content should be kept around 20% to minimize adsorption. 9
  10. 10. Why LCMS? 10
  11. 11. Major Techniques (LCMS, LBA) • Advantages of LCMS over LBA • Selectivity between similar peptides and proteins (quality information) • No antibodies required (less chances of cross reactivity) • Higher throughput (less time to develop methods and easy method transfer) • Simultaneous measurement of multiple analytes • High dynamic range (typically greater than 4 orders of magnitude) • Internal standard • Use of AC/LC-MSMS sensitivity can be greatly enhanced. • No reagent development. 11
  12. 12. 12
  13. 13. LCMS-Approach for peptide bioanalysis 13
  14. 14. Quantification through intact protein analysis. • This approach is the true measurement of whole protein. • MS uses SRM by Quadrapole and HRMS by Q-Tof or Orbitrap • HRMS provides high level of selectivity owing to the mass accuracy and sensitivity for some proteins which fragment poorly. • HRMS may also have advantage of quantifying proteins and their derivatives after biotransformation and post translation modifications. • Currently this approach is used for peptides <10kDa molecular weight. 14 Protein ------- Sample prep-------LCMS
  15. 15. Surrogate peptide approach • Commonly employed method for quantification of proteins. • Usually signature peptides which are unique to the protein are selected. • Selection Criteria: 1. Avoid peptides containing methionine, Cysteine or tryptophan 2. Avoid RR and KK in the sequence to minimize inconsistent digestion 3. Select peptides with length 5-15 amino acids.(too small too large may cause specificity and selectivity issues) • 4. Signature peptide can be generated by performing insilico digestion and can be searched against all proteins in the biological matrices. • https://www.expasy.org/proteomics • http://www.uniprot.org/ 15 Protein----digest-----peptides-----sample prep------LCMS
  16. 16. Protein digestion: • Different enzymes used for digestion (trypsin, Glu-C, Lys-C, chymotrypsin, Asp-N) • Trypsin is commonly employed owing to its availability, efficiency, low cost and generates smaller fragments which are MS friendly. • Conditions like incubation time, temperature, protein to enzyme ratio, organic content around 10-30% ACN to speed up the time can be explored for efficient and complete digestion. • Pretreatment with Urea, rapigest and guanidine HCl will enhance the digestion completeness. • Denaturation, reduction and alkylation are required for complete sequence coverage. • For quantitative bioanalysis direct digestion will also give suroogate peptides desired. 16
  17. 17. Sample clean up. • Plasma contains more than 60% of Albumin and 30—35% of immunoiglobulins. • Achieving analyte separation is essential to remove matrix effect or interfering peaks caused by wide isotopic abundance caused by endogenous proteins and peptides. • Most of the peptides and proteins contains mixture of aminoacids. Achieving sample clean up may be challenging. • Molecules with high polar aminoacids display weak solubility and with high % arginine residues will have a strong basic character. • SPE followed by PPT is a good approach to start with. 17
  18. 18. Sample clean Up Cont… • Generally larger protein precipitate around 40% of organic and smaller peptides may need higher % of organic solvent. • Aqueous acid preparations like 1% FA in ACN or 10% TFA in ACN can be tried if recovery is not consistent. • SPE avoids harsh organic conditions that can compromise solubility. • Lower limits of quantitation can be achieved. • Can be very selective with high recovery. • Size exclusion can also be used prior to SPE to remove larger proteins. • SPE needs expertise and with out SIL-IS chances of showing discrepancy in recovery is more. 18
  19. 19. Chromatography • For most LC seperations traditional C18 columns are good enough • Recent advances in column technology of sub 2µ particles with pore size of 300 A0 will provide increased resolution, sensitivity and speed. • Charge surface columns which will reduce secondary interactions are growing in popularity. • Decreasing flow rates to 200-400 µl/min, increasing column oven temperatures to 50 – 60 0C and performing shallower gradients will enhance sensitivity and peak shape. • Organic modifiers are always employed and acidic modifiers like formic acid and TFA can also be tried to neutralize carboxyl. • For very acidic and basic peptides or proteins ion exchange can also be tried. 19
  20. 20. Mass spectrometry. • Peptide and proteins generally multiply charged in ES source • Ratio of formation one charge state to other depends on flow rate and solvent composition. • So tuning to be performed with LC conditions. • Peptides and proteins demonstrate wider isotopic distributions limiting the sensitivity. • Monitoring several charge states and isotopes can help in increasing the sensitivity. • Digestion of peptides and monitoring surrogate peptides will also help • Peptides tend to fragment poorly. Increasing the collisional energy will compromise the selectivity as it leads to individual amino acids. • Avoid choosing immonium ions, water losses and adducts. • Good practice is to choose b or y ions for specificity. • With new technologies performing ETD will also generate more specific fragments like a and z ions. 20
  21. 21. Challenges using LCMS • Multiple charging and wide isotopic distribution restricts sensitivity • Flow rate and solvent will effect charge state and ionization • Poor fragmentation • m/z value may exceed instruments mass range. • Expertise • Lack of regulatory guidance. 21
  22. 22. Method validation 22
  23. 23. Method Validation cont…. • For LBS 4-6-20 rule applies(4 out of 6 should be within 20% of nominal value) • For LCMS 4-6-15 rule applies • Stability testing of digested proteins (false negatives if the peptide shows Non specific binding to wells, false positives if the peptide used is more stable than whole protein ) • Challenges during immunocapture purification with respect to IS. • All experiments needs to be performed as for small molecules like selectivity, precision and accuracy, stability studies, robustness and ruggedness. 23
  24. 24. Case Study Quantification of Peptide (Compound_T) in Plasma by HRMS (Orbitrap Fusion_Ultima 3000) 24
  25. 25. Blast to look for signature peptide • https://www.expasy.org/proteomics • http://www.uniprot.org/ 25
  26. 26. Signature peptide (Insilico digest) 26
  27. 27. Signature peptide (Blast) 27
  28. 28. Native form of the Peptide 201216_IP_01 #37 RT: 0.13 AV: 1 SB: 946 0.65-3.95 NL: 2.83E6 F: FTMS + p ESI Full ms [197.0777-1500.0000] 600 650 700 750 800 850 900 950 1000 1050 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 696.1371 z=6 835.1633 z=5 596.8327 z=7 1043.7020 z=4 635.3307 z=1 762.0109 z=6 914.4121 z=5 701.6351 z=6 845.9447 z=5 628.8601 z=? 663.3625 z=1 686.6325 z=6 607.2689 z=1 811.3070 z=? 770.8507 z=? 883.4208 z=? 948.2571 z=? 992.8575 z=5 829.5631 z=? 743.3185 z=? 715.3081 z=? 1035.7438 z=? Mass – 4171 Da Dissolved in 10% ACN in water (5ug/ml) 28
  29. 29. Reduced using DTT (600c for 30 min) 291216_TRIAL_IP_PEP_013 #1438 RT: 6.00 AV: 1 NL: 8.62E6 F: FTMS + p ESI Full ms [197.0777-1500.0000] 600 650 700 750 800 850 900 950 1000 1050 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 697.1456 z=6 836.3731 z=5 597.6971 z=7 1045.2148 z=4 706.1305 z=6 847.1561 z=5 687.6437 z=6 732.8215 z=6 824.7682 z=5 879.3820 z=5 762.6599 z=6 914.7878 z=5 974.5068 z=? 603.8462 z=7 660.8011 z=? 790.8429 z=2 1018.8467 z=? 947.5655 z=? Reduced mass – 4177 Da 29
  30. 30. Reduced and alkylated using Iodoacetamide 201216_BLK_05 #640 RT: 2.12 AV: 1 NL: 8.87E7 F: FTMS + p ESI Full ms [197.0777-1500.0000] 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 754.1687 z=6 646.5739 z=7 904.9985 z=5 1130.7469 z=4 565.8768 z=8 744.6634 z=6 761.1696 z=6638.4264 z=7 893.3944 z=5 1116.7415 z=4 913.0027 z=5 652.7184 z=7 1141.2510 z=4 735.1593 z=6 773.5038 z=6 613.9897 z=7 858.9816 z=5 1073.7217 z=4 580.3815 z=? 696.8102 z=? 829.5782 z=? 983.8463 z=? 1019.0507 z=? Fixed modification of CAM +57.02. Deconvuluted mass-4519 Da 30
  31. 31. Trypsin digest_peptide map(confirmation of sequence) RT: 0.00 - 35.00 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 Time (min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 15.78 11.82 1.32 13.20 15.97 16.20 16.8914.195.795.721.77 17.786.52 11.549.99 24.35 27.1520.28 22.98 31.9130.56 34.18 NL: 1.21E8 m/z= 246.1147-246.1197+303.1474-30 368.6723-368.6797+408.6503-40 463.2108-463.2200+573.2018-57 F: FTMS + p ESI Full ms [197.077 201216_PEPMAP_TRYDIGEST_ Sequence- DCLGWFKGCD PDNDKCCEGY KCNRRDKWCK 2-17,9-22,46-29 disulfide linkages PEPTIDE MASS MODIFICATION PEPTIDE SEQUENCE 859.3162 9,16,17 GCDPDNDKCCEGYK 463.2154 2 DCLGWFK 460.6743 9 GCDPDNDK 408.6544 16,17 CCEGYK 368.676 29 DKWCK 305.1734 YKLW 303.1504 22 CNRR Digestion in 10% ACN for 4 hrs at 370c 31
  32. 32. MSMS spectra for peptide(DCLGWFK)- 463.2154(M+H)2+ 080117_ext_TRIAL_041 #1886 RT: 3.95 AV: 1 SB: 245 3.37-3.85 , 4.05-5.09 NL: 1.45E5 F: FTMS + c ESI Full ms2 463.2154@hcd25.00 [100.0000-1200.0000] 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 650.3652 537.2812 463.1800 276.0645 133.0608 294.1808 248.0697 147.1128 357.1917 504.2860159.0764 448.1631 302.1134 405.7023 234.0541 820.3802336.2276 476.2634 630.4291 755.4230173.4111 712.4088561.3227 766.3542 668.3898 PEPTIDE SEQUENCE M/Z DC LGWFK 650.3652 DCL GWFK 537.2812 DCLG WFK 480.2597 DCLGW FK 294.1808 DCLGWF K 147.1128 IMMONIUM IONS 129.1023(k) 159.0764(W) 120.0804(F) characteristic mass presence of k in the sequence 32
  33. 33. MSMS spectra for peptide(YKLW)- 305.1734(M+H)2+ 080117_ext_TRIAL_041 #1858 RT: 3.89 AV: 1 SB: 244 3.37-3.85 , 4.05-5.09 NL: 1.99E5 F: FTMS + c ESI Full ms2 305.1734@hcd25.00 [100.0000-1200.0000] 100 150 200 250 300 350 400 450 500 550 600 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 136.0757 129.1022 188.0705 446.2754 305.1567 205.0970159.0916119.0492 242.1861 318.1807 292.1652 405.2495270.1812224.1753 428.2649387.2387 360.2266 468.1993 550.9755 590.5245492.5654 609.6941 328.7218182.5009 PEPTIDE SEQUENCE M/Z Y KLW 446.2754 YK LW 318.1807 YKL W 205.097 IMMONIUM IONS 129.1023(K) 136.0757(Y) 159.0916(W) 33
  34. 34. Blank and LLOQ (1 ng/ml) of two unique peptides RT: 0.00 - 8.00 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 Time (min) 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 3.85 3.93 3.94 4.01 1.29 NL: 2.07E4 m/z= 446.2736-446.2780 F: FTMS + c ESI Full ms2 305.1734@cid25.00 [100.0000-1200.0000] MS 080117_ext_TRIAL_029 NL: 3.21E4 m/z= 650.3630-650.3696 F: FTMS + c ESI Full ms2 463.2154@cid25.00 [100.0000-1200.0000] MS 080117_ext_TRIAL_029 NL: 8.83E2 m/z= 446.2736-446.2780 F: FTMS + c ESI Full ms2 305.1734@cid25.00 [100.0000-1200.0000] MS 080117_ext_trial_001 NL: 0 m/z= 305.1719-305.1749 F: FTMS + c ESI Full ms2 463.2154@cid25.00 [100.0000-1200.0000] MS 080117_ext_trial_001 Lloq- 1 ng/ml Plasma BLK 34
  35. 35. Chromatograms of different concentrations for peptide YKLW RT: 0.00 - 8.00 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 Time (min) 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 1.29 3.85 3.93 3.83 3.93 3.87 3.95 3.83 3.91 4.02 NL: 8.83E2 m/z= 446.2736-446.2780 F: FTMS + c ESI Full ms2 305.1734@cid25.00 [100.0000-1200.0000] MS 080117_ext_trial_001 NL: 2.07E4 m/z= 446.2736-446.2780 F: FTMS + c ESI Full ms2 305.1734@cid25.00 [100.0000-1200.0000] MS 080117_ext_TRIAL_029 NL: 5.27E4 m/z= 446.2736-446.2780 F: FTMS + c ESI Full ms2 305.1734@cid25.00 [100.0000-1200.0000] MS 080117_ext_trial_030 NL: 1.17E5 m/z= 446.2736-446.2780 F: FTMS + c ESI Full ms2 305.1734@cid25.00 [100.0000-1200.0000] MS 080117_ext_trial_031 NL: 1.89E5 m/z= 446.2736-446.2780 F: FTMS + c ESI Full ms2 305.1734@cid25.00 [100.0000-1200.0000] MS 080117_ext_TRIAL_032 1 ng/ml 2.5 ng/ml blk 5ng/ml 10 ng/ml 35
  36. 36. Points to consider • pH to be maintained at around 8 for Activity of DTT and Trypsin • Percentage of organic modifier can be tried for enhanced digestion • Sample were stored in -200c and were found to be stable for 14 days • Ice bath was used during processing and temperature was maintained around 4 in centrifugal SPE. • Oasis MAX was used as sorbent for sample clean up • Protein heterogenicity to be considered (PTMs) and modifications during processing. • Calculation of net charge will also help in deciding SPE chemistry. 36
  37. 37. Conclusion • Quantification using HRMS for proteins and large molecules gives better selectivity and sensitivity. • HRMS gives qualitative and quantitative data. • Modifications can be detected. • Intact protein bioanalysis • Less time for development of analytical method. • Depleting high abundance proteins can increase the sensitivity. 37
  38. 38. References • http://www.ionsource.com/ • https://www.expasy.org/proteomics • Therapeutic peptide bioanalysis_Sciex application note. • Peptide and protein bioanalysis_Waters application note. • Quantitative bioanalysis of proteins by mass spectrometry. Long Yuan et al. • Approaches to analyzing therapeutic peptides and proteins by LC-MS/MS. Matthew Ewles • LC-MSMS of large molecule in regulated bioanalytical environment-which acceptance criteria to apply. Magnus Knotssun. • Protein and peptide drug analysis by MS: Challenges and oppurtunities for the discovery environment. J.larry CAmpbell • LC-MS-based bioanalysis in support of protein biotherapeutics development: • current challenges & emerging opportunities. Rand Jenkins 38
  39. 39. Acknowledgements • Mrs. Huda Zaheer • Mrs. Amrita Rajagopal • Mr. Aazam Syed • Dr. Dhanapal swaminathan 39
  40. 40. Large Meets Small Thank You 40

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