2007 Waters Corporation. Application of UPLC to Peptide Mapping
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1 2007 Waters Corporation Application of UPLC to Peptide Mapping
2 Comparison of HPLC and UPLC 7.5e-2 7.0e-2 6.5e-2 6.0e-2 5.5e-2 HPLC 5.0e-2 4.5e-2 4.0e-2 3.5e-2 3.0e-2 2.5e-2 2.0e-2 1.5e-2 1.0e e-2 8.0e-2 7.0e-2 UPLC 6.0e-2 5.0e-2 4.0e-2 3.0e-2 2.0e Waters Corporation 2
3 Magnified Comparison HPLC and UPLC 7.5e-2 7.0e-2 6.5e-2 6.0e-2 5.5e-2 5.0e-2 4.5e-2 HPLC 4.0e-2 3.5e-2 3.0e-2 2.5e-2 UPLC 2.0e Waters Corporation 3
4 Comparison of HPLC and UPLC Constant L/dp and Gradient 7.5e-2 7.0e-2 6.5e-2 6.0e-2 5.5e-2 5.0e-2 HPLC Peak Capacity = e-2 4.0e-2 3.5e-2 3.0e-2 2.5e-2 2.0e-2 1.5e-2 1.0e e-2 8.0e-2 7.0e-2 6.0e-2 5.0e-2 UPLC Peak Capacity = e-2 3.0e-2 2.0e Waters Corporation 4
5 Uses of Peptide Mapping Structural Characterization Pattern conforms to primary structure Used with MS for primary structure determination Protein Modification Identify post-translational modifications o Glycosylation, substitution, truncation Determine product related impurities o Deletions, deamidation, oxidation, etc. Protein Identity Establish product identity by confirming primary structure Confirm presence of signature peptides Product integrity lot-to-lot analysis 2007 Waters Corporation 5
6 UltraPerformance LC A new class of separation science Based on chromatography columns with very small particles Based on instruments designed to take advantage of the small particles Provides improved Resolution, Speed, and Sensitivity with no compromises Suitable for chromatographic applications in general Appropriate for developing new methods Appropriate for improving existing methods 2007 Waters Corporation 6
7 Application of UPLC to Peptide Mapping Special Considerations Large molecules Diffusion is slow Available surface Polydisperse with respect to size and shape Wide range of chemical properties Small chemical differences between large molecules 2007 Waters Corporation 7
8 Application of UPLC to Peptide Mapping Transferring HPLC methods Considerations for optimization Flow rate/linear velocity Gradient slope Mobile phase modifiers Modified peptides Quantitation Future Directions 2007 Waters Corporation 8
9 Transferring A Peptide Separation HPLC to UPLC N L dp Efficiency, N, is directly proportional to column length, L, and inversely proportional to particle size, dp 2007 Waters Corporation 9
10 Comparison of HPLC and UPLC Constant L/dp and Gradient 7.5e-2 7.0e-2 6.5e-2 6.0e-2 5.5e-2 HPLC 5.0e-2 4.5e-2 4.0e-2 3.5e-2 3.0e-2 2.5e-2 2.0e-2 1.5e-2 1.0e e-2 8.0e-2 7.0e-2 UPLC 6.0e-2 5.0e-2 4.0e-2 3.0e-2 2.0e Waters Corporation 10
11 Comparison of HPLC and UPLC Scaled Gradient 7.0e-2 6.0e-2 5.0e-2 HPLC 1.5%/CV 4.0e-2 3.0e-2 2.0e-2 90min 1.0e e-1 9.0e-2 8.0e-2 7.0e-2 6.0e-2 5.0e-2 UPLC 1.5%/CV 4.0e-2 3.0e-2 2.0e-2 55min Waters Corporation 11
12 Effect of Column Length Scaled Gradient 1.0e-1 8.0e-2 6.0e-2 150mm 55min 4.0e-2 2.0e-2 1.4e-1 1.2e-1 1.0e-1 8.0e-2 6.0e-2 4.0e mm 38min 2.0e e-2 5.0e-2 4.0e-2 3.0e-2 50mm 20min 2.0e Waters Corporation 12
13 Transfer to Longer Column 2.1 x 50 % x 150 % Waters Corporation 13
14 Linear and Segmented Gradients Gradient Slopes %B 30 Initial 0.5%/cv Segment Waters Corporation 14
15 Segmented Gradient 6.0e-2 4.0e-2 2.0e %/cv 6.0e e-2 2.0e %/cv 6.0e-2 4.0e-2 2.0e Waters Corporation %/cv
16 Fast, Focused Analysis 1.4e-1 1.3e-1 1.2e-1 1.1e-1 1.0e-1 9.0e-2 8.0e-2 7.0e-2 6.0e-2 5.0e-2 4.0e-2 3.0e-2 2.0e-2 1.0e Waters Corporation 16
17 Application of UPLC to Peptide Mapping Transferring HPLC methods Considerations for optimization Flow rate/linear velocity Gradient slope Mobile phase modifiers Modified peptides Quantitation Future Directions 2007 Waters Corporation 17
18 Chromatography Principles Mass Transfer / Diffusion Mobile Phase Analyte molecules Porous Particle Diffusion-related band broadening Adsorption Equilibria 2007 Waters Corporation 18
19 Effect of Flow Rate Peptide Standard μL/min Peak 1 300μL/min 33μL 100μL/min 20μL μL 33μL % μL 24μL 25μL 19μL μL 18μL μL/min μL 30μL 36μL 18μL 19μL 20μL μL 18μL % μL 24μL Higher Concentration Waters Corporation 19
20 Chromatography Principles Mass Transfer / Diffusion Mobile Phase Analyte molecules A B Porous Particle C 2007 Waters Corporation 20
21 van Deemter Plot Small Molecule H (cm) micron particles small molecules 1.7 micron particles small molecules u (mm/sec) 2007 Waters Corporation 21
22 van Deemter Plot 1500da Peptide H (cm) 3.5 micron particles peptides 1.7 micron particles peptides u (mm/sec) 2007 Waters Corporation 22
23 van Deemter Plots Small Molecules Peptides H (cm) H (cm) u (mm/sec) u (mm/sec) 3.5μm particles 1.7μm particles 2007 Waters Corporation 23
24 van Deemter Plot 1500da Peptide μL/min 2.1mm Column 250μL/min 2.1mm Column H (cm) 3.5 micron particles peptides 1.7 micron particles peptides u (mm/sec) 2007 Waters Corporation 24
25 UPLC Peptide Mapping 100mL/min Retention Reproducibility 29.52± ± ± ±.024 % 3.54± Waters Corporation 25
26 Effect of Flow Rate e-2 5.0e-2 4.0e-2 3.0e-2 2.0e-2 1.0e μL/min e-2 5.0e-2 4.0e-2 3.0e-2 2.0e-2 1.0e μL/min Waters Corporation 26
27 Application of UPLC to Peptide Mapping Transferring HPLC methods Considerations for optimization Flow rate/linear velocity Gradient slope Mobile phase modifiers Modified peptides Quantitation Future Directions 2007 Waters Corporation 27
28 Effect of Gradient Slope Constant Flow Rate Phosphorylase Standard BEH,1.7u;2.1x50;0.1mL/min; 0-50, 29min; 3.0%/cv 1.5e e-1 1.0e-1 3.0%/cv 7.5e-2 5.0e-2 2.5e Phosphorylase Standard BEH,1.7u;2.1x50;0.1mL/min; 0-50, 58min; 1.5%/cv 1.2e-1 1.0e-1 8.0e-2 1.5%/cv 6.0e-2 4.0e-2 2.0e Waters Corporation 28
29 Method ptimization: Gradient Slope Rate of Change 0.75%/ col. vol % % * * * 1 Rate of Change 1.5%/ col. vol Waters Corporation 29
30 Effect of Gradient Slope Shallower gradient slope may improve resolution Decreasing gradient slope will decrease sensitivity Steeper gradient slope may compress the peaks and often reduce the resolution Increasing gradient slope will increase sensitivity Changing gradient slope is a balance between peak heights relative to resolution Changes in retentivity and selectivity 2007 Waters Corporation 30
31 Application of UPLC to Peptide Mapping Transferring HPLC methods Considerations for optimization Flow rate/linear velocity Gradient slope Mobile phase modifiers Modified peptides Quantitation Future Directions 2007 Waters Corporation 31
32 RP Retention of Peptides Non-Polar Surface (+) Si Si Si Si H H CH 3 Si Si (CH 2 ) 17 CH 3 CH 3 Si H CH 3 Si (CH 2 ) 17 CH 3 CH 3 CH 3 Si CH 3 CH 3 (-) Multiple Charges result in very little attraction to particle surface 2007 Waters Corporation 32
33 RP Retention of Peptides Non-Polar Surface (+) Si Si Si Si H H CH 3 Si Si (CH 2 ) 17 CH 3 CH 3 Si H CH 3 Si (CH 2 ) 17 CH 3 CH 3 CH 3 Si CH 3 CH 3 Lowering the ph of mobile phase results in protonation of acidic site H Reduction in # of Charged sites results in an increased attraction to particle surface 2007 Waters Corporation 33
34 RP Retention of Peptides Non-Polar Surface Si Si Si Si H H CH 3 Si Si (CH 2 ) 17 CH 3 CH 3 Si H CH 3 Si (CH 2 ) 17 CH 3 CH 3 CH 3 Si CH 3 CH 3 (+)(-)TFA Reduction in all Charged sites results in greatest attraction to particle surface Using TFA, which lowers the ph AND has an IN PAIRING capability for the basic site, results in no net charges on the surface Lowering the ph of mobile phase results in protonation of acidic site H 2007 Waters Corporation 34
35 Effect of Mobile Phase Modifier in UPLC ACQUITY UPLC Column 100 % FA % TFA Waters Corporation 35
36 Peptide Separation Technology Flexible Choice of Mobile Phase Modifier % 0 % 5 0.1% FA % TFA Waters Corporation 36
37 Formic Acid vs TFA Effect on UV Sensitivity TFA 2.4e-2 2.2e-2 2.0e-2 1.8e-2 1.6e-2 1.4e-2 1.2e-2 1.0e-2 8.0e-3 6.0e-3 4.0e-3 2.0e FA 5.0e-2 4.5e-2 4.0e-2 3.5e-2 3.0e-2 2.5e-2 2.0e-2 1.5e-2 1.0e-2 5.0e Waters Corporation 37
38 Application of UPLC to Peptide Mapping Transferring HPLC Methods Considerations for optimization Flow rate/linear velocity Gradient slope Mobile phase modifiers Protein Glycosylation Quantitation Future Directions 2007 Waters Corporation 38
39 IgG Structure 2007 Waters Corporation 39 NH 2 NH2 S S S S S S S S S S S S S S S S V H C H 1 S S S S C H 2 S S S S C H 3 S S S S S S S S V L C C H 1 L C H 2 C H 3 C L V H V L CH C H CH CH NH NH 2 2 Glycans MW ~ 147,000
40 Neutral N linked N Glycopeptides Tryptic Digest of Monoclonal Mouse IgG IgG Glycopeptides 1.0e-2 UV 8.0e % 6.0e-3 4.0e-3 2.0e TIC Waters Corporation 40
41 Neutral N-linked N Glycopeptides EEQFNSTFR = Peptide Sequence G0 G1 G Galactose N-Acetyl glucosamine Mannose Fucose 2007 Waters Corporation 41
42 Neutral N N linked Glycopeptides % G2 (M+H) % G1 (M+H) % G0 (M+H) Mouse IgG glycoforms were detected by extracted ion chromatograms of the triply-chargedglycopeptides are shown. Four peaks were observed for the three molecular weights indicating that the positional isomers of G1 have been separated by UPLC Waters Corporation 42
43 Neutral N N linked Glycopeptides G1-containing glycopeptide isomers 6 F N 6 F N 3 S 3 S T T Galactose N-Acetyl glucosamine Mannose Fucose 2007 Waters Corporation 43
44 IgG Glycopeptide UPLC 1.9e UV 1.8e-2 1.7e-2 1.6e-2 1.5e e-2 1.3e e-2 1.1e-2 1.0e-2 9.0e Waters Corporation 44
45 Application of UPLC to Peptide Mapping Transferring HPLC methods Considerations for optimization Flow rate/linear velocity Gradient slope Mobile phase modifiers Modified peptides Quantitation Future Directions 2007 Waters Corporation 45
46 Quantitative Analysis in Peptide UPLC Experimental Design MassPREP Peptide Standard Measure retention time reproducibility Measure changes in resolution with amount Measure linearity of peak area with amount Spike standard into Hemoglobin digest Select surrogate peak Measure retention reproducibility Measure linearity of surrogate peak area Measure detectable peptide as percentage of digest 2007 Waters Corporation 46
47 Peptide Quantitation Retention time Reproducibility e-2 7.0e e e-2 4.0e-2 3.0e-2 2.0e Waters Corporation 47
48 Peptide Quantitation Linearity 2.75e-1 2.5e e-1 2.0e e-1 1.5e e-1 1.0e-1 7.5e-2 5.0e-2 2.5e pmol 2007 Waters Corporation 48
49 Peptide Quantitation Linearity 2.6e-2 2.4e-2 2.2e-2 pmol 2.0e-2 1.8e-2 1.6e-2 1.4e Waters Corporation 49
50 Peptide Map Trace Contaminant e e-1 1.0e e-2 * Waters Corporation 50
51 Peptide Map Trace Contaminant 1.4e e-1 1.3e e-1 1.2e e-1 1.1e e-1 1.0e-1 9.5e-2 9.0e-2 8.5e-2 8.0e-2 7.5e-2 7.0e-2 6.5e-2 6.0e-2 5.5e-2 5.0e-2 4.5e-2 4.0e-2 3.5e-2 3.0e-2 2.5e-2 2.0e-2 * % 1% 0.5% 0.2% 2007 Waters Corporation 51
52 Application of UPLC to Peptide Mapping Transferring HPLC Methods Considerations for optimization Flow rate/linear velocity Gradient slope Mobile phase modifiers Modified peptides Quantitation Future Directions 2007 Waters Corporation 52
53 BEH Technology Bridged EthylSiloxane/Silica Hybrid Particles Et 4 Si Et Et Et Tetraethoxysilane (TES) Et Et + Si 1 Et CH 2 CH 2 Si Et Et Et Bis(triethoxysilyl)ethane (BTEE) Et Et Si Si CH 2 CH 2 Si Si Et Et Si Si Polyethoxysilane (BPES) Et Et Et Et n Bridged Ethanes In Silica Matrix 2007 Waters Corporation 53
54 Peptide Separation Technology ACQUITY UPLC Columns for Peptide Mapping Based on the C18 1.7um BEH Particles Can be used with TFA and Formic Acid modifiers Available in 130Å and 300Å Specifically QC tested with a peptide map 2007 Waters Corporation 54
55 Columns for UPLC Peptide Mapping Peptide Separation Technology ACQUITY UPLC columns QC tested with peptide maps ACQUITY UPLC BEH130 C18 1.7µm, 2.1mm x 50/100/150mm ACQUITY UPLC BEH300 C18 1.7µm, 2.1mm x 50/100/150mm 2007 Waters Corporation 55
56 Tryptic Digest of Phosphorylase b A 4 residue e-1 1.0e-1 B 25 residue C 20 residue A B C ACQUITY BEH 130Å 8.0e-2 6.0e-2 4.0e-2 2.0e e-2 5.0e B A C ACQUITY BEH 300Å 4.0e-2 3.0e-2 2.0e-2 1.0e Waters Corporation 56
57 Lys-C C Digest of Phosphorylase b A 4 residue e-1 1.6e-1 1.4e-1 1.2e-1 1.0e-1 8.0e-2 6.0e-2 B 25 residue C 36 residue A B C ACQUITY BEH 130Å 4.0e-2 2.0e e-1 1.6e-1 1.4e-1 C ACQUITY BEH 300Å 1.2e-1 1.0e-1 8.0e-2 A B 6.0e-2 4.0e-2 2.0e Waters Corporation 57
58 UPLC for Peptide Mapping Summary Useful for tryptic digests, even larger than 40 residues Improves resolution compared to HPLC Similar, not identical selectivity Best resolution at low linear velocity/ flow rate Best resolution with shallow gradients Desirable Separation Chemistry Good separation with formic acid Good peak shape with glycopeptides Improves sensitivity Enhanced resolution reveals trace components Reduced peak volume increases detector response 2007 Waters Corporation 58
59 UPLC Peptide Mapping Application Solution Complete UPLC Systems with UV and MS Detection Higher resolution for protein identification and easier variant recognition Increased sensitivity and dynamic range for quantitation of trace peptides Faster run times for throughput and efficient methods development Reduce consumption of precious sample and costly reagents Application-specific system PQ Unique Peptide Separations Technology Columns Exceptional chromatographic performance Column QC test with protein digest map for ensured reproducibility Common Applications Peptide mapping for biopharmaceuticals Characterization of synthetic peptides 2007 Waters Corporation 59
60 Evaluation Form We Appreciate your pinion Please complete Evaluation Form Qualify for drawing to win a $25 AMEX gift card (drawn at end of this session must be present to win) AND Qualify to win a Gold Package registration to Inform 2007 ($1595 value; one winner per day; winner to be notified post-pittcon) Thank You! 2007 Waters Corporation 60
61 Inform 2007 Where is it located and how do I register Waters premier Laboratory Informatics symposium where users can network with colleagues, share experiences, and gain valuable insights into how to best deploy and use Waters Informatics suite of solutions May 7-10, 2007 Miami Marriott Biscayne Bay Three-day event with optional tutorials Monday: (2) ½ Day Tutorial Session Tuesday Thursday: Symposium Download the agenda and register at: Waters booth Fill out a seminar evaluation form and enter the raffle to win a Gold Package registration* for Inform 2007 which includes all tutorials and workshops - a $1595 value ne drawing daily. Winner will be notified. * Prize covers conference registration only 2007 Waters Corporation 61
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