Practical Method Development and Optimization for UHPLC and HPLC Separations of Peptides Practical Method Development and Optimization for UHPLC and HPLC Separations of Peptides for Peptide Mapping Analyses Using Different Mobile Phase Additives for Peptide Mapping Analyses Using Different Mobile Phase Additives Thomas J. Waeghe 1 , Benjamin Libert 2 , Stephanie A. Schuster 2 , and Barry E. Boyes 2 Thomas J. Waeghe , Benjamin Libert , Stephanie A. Schuster , and Barry E. Boyes 1 MAC MOD Analytical Inc 103 Commons Court Chadds Ford PA 19317; 2 Advanced Materials Technology Inc Wilmington DE 19810 Presented at EAS 2016 1 MAC-MOD Analytical, Inc., 103 Commons Court, Chadds Ford, PA 19317; 2 Advanced Materials Technology Inc., Wilmington, DE 19810 Presented at EAS 2016 Abstract Parameters That Affect Peptide LC MS Separations of Enolase Tryptic Digest LC MS Extracted Ion Chromatograms Total-ion Chromatogram Showing Enolase Tryptic Digest Abstract Reversed-Phase Gradient Separations LC-MS Separations of Enolase Tryptic Digest Using Different Mobile Phase Additives LC-MS Extracted-Ion Chromatograms for Gradient Input Runs at Two Temperatures PTGAK LIVDAIK Total ion Chromatogram Showing Enolase Tryptic Digest Fragments Obtained Using Near-Optimum Conditions Parameter Determined by Impacts performance Using Different Mobile Phase Additives for Gradient Input Runs at Two Temperatures 9.0e6 EMR LR SHFFK QEFMIAP AEEALDL TIC Tune File Values Source Type: HESI Peptide mapping is a critical competency and technique for both research and development and for quality assurance and product release for many new Parameter Determined by Impacts performance • Most labs choose acetonitrile for viscosity • Peak width Same LC conditions as shown on previous panel for LC-UV 10 mM Trifluoroacetic Acid 20 mM Formic Acid 2.5 0.90 0.95 1.00 (x1,000,000) 1:626.30(+) 1:597.30(+) 1:572.30(+) 1:560.30(+) 1:550.80(+) 1:546.30(+) 1:404.20(+) 1:387.20(+) 1:378.70(+) 1:373.20(+) 15.121 14.426 15.502 30 min 0.90 0.95 1.00 (x1,000,000) 1:626.30(+) 1:597.30(+) 1:572.30(+) 1:560.30(+) 1:550.80(+) 1:546.30(+) 1:404.20(+) 1:387.20(+) 1:378.70(+) 1:373.20(+) 26.625 60 min 8.0e6 HGDKL VHEALE TANK MK DLVVGL DWEAWS AGGALALQ VAPNIQTA Note: MS spectra were acquired with data-dependent acquisition Capillary Temp (C): 225.00 Source Heater Temp (C): 150.00 Sheath Gas Flow (): 35.00 Aux Gas Flow (): 10 00 development and for quality assurance and product release for many new biopharmaceuticals including peptide drugs, monoclonal antibody drugs and Organic modifier • Most labs choose acetonitrile for viscosity, mass transfer, efficiency reasons, UV transparency, ionization efficiency in MS • Selectivity • Retention • Peak capacity chromatograms MS Scan: 0 2 s (300–2000 m/z) 2.0 0.65 0.70 0.75 0.80 0.85 1:1247.30(+) 1:995.50(+) 1:921.50(+) 1:911.50(+) 1:896.50(+) 1:889.40(+) 1:878.50(+) 1:814.40(+) 1:807.90(+) 1:789.90(+) 1:733.40(+) 1:708.70(+) 1:707.15(+) 1:702.20(+) 1:659.40(+) 1:644.90(+) 10.382 11.920 19.519 30 min 60 C 0.65 0.70 0.75 0.80 0.85 1:1247.30(+) 1:995.50(+) 1:921.50(+) 1:911.50(+) 1:896.50(+) 1:889.40(+) 1:878.50(+) 1:814.40(+) 1:807.90(+) 1:789.90(+) 1:733.40(+) 1:708.70(+) 1:707.15(+) 1:702.20(+) 1:659.40(+) 1:644.90(+) 24 35.205 25.506 27.324 60 min 60 C 7.0e6 K DK TTEK GENFHH GASTGV K 5] SHR ISLDGT DLYHSLM EDTFIAD PFAEDD SHFFK LNGGSHA VGDEGGV Aux Gas Flow (): 10.00 POSITIVE POLARITY Source Voltage (kV): 2.20 Source Current (uA): 100.00 antibody-drug conjugates (ADCs). New 2-μm superficially porous columns, capable of pressures up to 1000 bar can deliver high resolution /peak capacity • Peak capacity Mobile phase • UV transparency • Peak shape • Peak width MS Scan: 0.2 s (300–2000 m/z) Multiple Ion Chromatogram scale (x 10 6 ) 1.5 0.40 0.45 0.50 0.55 0.60 1:1629.30(+) 10.780 0.600 12.271 11.066 12.818 0.40 0.45 0.50 0.55 0.60 1:1629.30(+) 17.453 18.22 38.094 .420 4 22.723 2 5 0e6 6.0e6 AIEK TAIEK VYHNLK KNPNS DK TVEVELT NAVFAG SIVPSG PAFVK DLTVTNPK methyl [C5 GWGVMVS AVDDFL GPQLAD SGETE VSIEDP WEAWS PVPFLNVL GASAGNV S-Lens RF Level (%): 69.00 FTMS Full Micro Scans: 1 FTMS Full Max Ion Time (ms): 50.00 capable of pressures up to 1000 bar, can deliver high resolution /peak capacity separations, and can be used at temperatures up to 60 C. Moreover, a variety additive type and concentration UV transparency • LC-MS sensitivity (S/N) • Column stability at pH and temperature Peak width • Loading (dynamic range) • Selectivity LC MS i l All chromatogram are on the same scale. 1.0 0.15 0.20 0.25 0.30 0.35 4.721 9 .354 13.497 8.634 13.747 0 9.066 882 3 8.900 10.747 438 015 0 390 23.699 13.766 14.430 6 5.880 7.930 0.15 0.20 0.25 0.30 0.35 25.868 23.390 24.098 2 372 2.647 1. 13.948 75 34.880 4 21.604 24.113 25.511 18.110 55 3.493 4.0e6 5.0e6 SK IATA RIAT AVK IGSEV KYDLDF LR KNPNSD GNPT ALLLK EELGDN VNDVIAP GIQIVADD arbamidom QDSFAAG R A WLTG YPIV FAEDDW TSPYVLP min) RYG LLR LSK Data-Dependent Acquisition Settings Scan Event Details: 1: FTMS + positive high res=30000 of mobile phase additives include trifluoroacetic acid (TFA), ammonium formate/formic acid buffers and a new additive, difluoroacetic acid (DFA), can • LC-MS signal Gradient steepness • User experience S l l it • Selectivity P k it 0.5 1.00 (x1,000,000) 1:37870(+) 1:373.20(+) 662 5 1.00 (x1,000,000) 1:37870(+) 1:373.20(+) 5 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 min 0.00 0.05 0.10 0.15 2.513 14. 15.11 5. 18. 2.250 7.8 12.973 6.4 24.0 8.307 23.3 19.492 7.373 21.873 9.076 15 9.416 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 55.0 57.5 min 0.00 0.05 0.10 0.15 17.193 2 17.193 25.895 9.893 11.793 2.262 46.940 1.077 14.3 12 15.033 15.907 9.475 29.653 44.164 2 16.200 28.05 3.0e6 AR DSR DVK HLADLS GVLHA DGK TFAEAL YDLDFK AADA LSESIK IEE NV TAG SEFFK C AAQ GVSLAAS VSIEDPF T (19.7 LNQ K KHLADL (200.0–2000.0) Collision Voltage = 0.0V 2: ITMS + p norm Dep MS/MS formate/formic acid buffers and a new additive, difluoroacetic acid (DFA), can be applied for effective development and optimization of rugged and robust separations of comple en me digests Gradient steepness • Sample complexity • Experimentation and optimization software • Peak capacity • Peak height • Selectivity 00 50 10 0 15 0 20 0 25 0 30 0 35 0 min 00 50 10 0 15 0 20 0 25 0 30 0 35 0 min 0.0 Comparison of multiple-ion f C S 0.75 0.80 0.85 0.90 0.95 1:80790(+) 1:789.90(+) 1:733.40(+) 1:708.70(+) 1:707.15(+) 1:702.20(+) 1:659.40(+) 1:644.90(+) 1:626.30(+) 1:597.30(+) 1:572.30(+) 1:560.30(+) 1:550.80(+) 1:546.30(+) 1:404.20(+) 1:387.20(+) 1:378.70(+) 15.66 913 20.691 16.615 30 min 30 C 0.75 0.80 0.85 0.90 0.95 1:80790(+) 1:789.90(+) 1:733.40(+) 1:708.70(+) 1:707.15(+) 1:702.20(+) 1:659.40(+) 1:644.90(+) 1:626.30(+) 1:597.30(+) 1:572.30(+) 1:560.30(+) 1:550.80(+) 1:546.30(+) 1:404.20(+) 1:387.20(+) 1:378.70(+) 27.945 27.984 37.956 60 min 30 C 2.0e6 TGAPA SVYD ANID T Y NQIGTL IGLDCAS LGANAILG RYPIV AADALLLK NVPLYK 2: ITMS + p norm Dep MS/MS (110.0–2000.0) Most intense ion from (1) separations of complex enzyme digests. Column temperature • Analyte requirements (recovery, stability) • Column stability requirements • Flow rate (thermal heating) • Selectivity • Recovery • Analysis time 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 min 10 mM Difluoroacetic Acid 10 mM Ammonium Formate 10 mM Formic Acid 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 min chromatograms from LC-MS analyses of enolase tryptic digests using formic acid ammonium 050 0.55 0.60 0.65 0.70 1:1629.30(+) 1:1247.30(+) 1:995.50(+) 1:921.50(+) 1:911.50(+) 1:896.50(+) 1:889.40(+) 1:878.50(+) 1:814.40(+) 1:807.90(+) 11.332 11.9 13.487 15.049 133 30 C 0.55 0.60 0.65 0.70 1:1629.30(+) 1:1247.30(+) 1:995.50(+) 1:921.50(+) 1:911.50(+) 1:896.50(+) 1:889.40(+) 1:878.50(+) 1:814.40(+) 1:807.90(+) 19.512 20.672 26.955 9.726 30 C 1.0e6 VN L KA N Activation Type: CID Min. Signal Required: 500.0 Isolation Width: 4.00 Normalized Coll Energy: 35 0 We will demonstrate the usefulness of these columns and additives in the • Flow rate (thermal heating) • Instrument capability and pressure limit • Peak width • Peak capacity Note: 2.2–52.2% in 40 min using formic acid, ammonium formate/formic acid, trifluoroacetic acid, and difluoroacetic acid as 0.30 0.35 0.40 0.45 0.50 14.052 15.349 10.107 7 12.068 14. 0.30 0.35 0.40 0.45 0.50 19.553 27.496 23 48.007 24.226 20.291 25.507 29 0.0e0 Normalized Coll. Energy: 35.0 Default Charge State: 2 Activation Q: 0.250 Activation Time: 30.000 CV 0 0V development of peptide mapping separations, and will offer a stepwise method development and optimization strategy for such separations. Flow rate • User choice • Column and Instrument pressure limits C • Selectivity • Peak capacity mobile phase additives. DFA offered 2 3 fold better signal 0.05 0.10 0.15 0.20 0.25 15.369 6.381 17.407 1 3.374 1.554 11.104 3.260 8.707 10.601 7.289 26.001 23.046 23.419 15.367 15.037 4.267 10.604 0.05 0.10 0.15 0.20 0.25 27.430 52.713 36.687 9.243 24.719 33.113 7.120 10.653 2 44.340 7.380 3.638 17.510 15.443 16.42 11.196 45.171 43.241 43.965 2.813 11.007 24.820 30.281 51.580 27.476 27.932 36.820 26.950 27.000 41.907 2.687 12.586 21.547 30.569 18.346 2 4 6 8 10 12 14 16 18 -1.0e6 min CV = 0.0V Number of Scan Events: 6 86% Coverage: Enolase 1 OS=Saccharomyces cerevisiae (strain ATCC 204508 / S288c) GN=ENO1 PE=1 SV=3 development and optimization strategy for such separations. • Column particle morphology and diameter Peak capacity • Packing particle size DFA offered 2–3 fold better signal intensity compared to TFA for LC- MS, with comparable narrow peaks 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 min 0.00 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 55.0 57.5 min 0.00 Sample: Enolase Tryptic Digest in 0.5% HCOOH, 2 L of 1 μg/μL Column: HALO 2 Peptide ES-C18, 2.1 x 100 mm, 2 m Flow: 0 3 mL/min Instrument: Shimadzu Nexera UHPLC MS: Thermo Orbitrap Velos Pro ETD Pressure Packing particle size • Column length • Flow rate Column temperature • Retention • Selectivity MS, with comparable narrow peaks and retention. For LC analysis conditions, see panel 8 Flow: 0.3 mL/min Mobile Phase A: 0.1% (v/v) DFA in water Mobile Phase B: 0.1% (v/v) DFA in CH 3 CN Gradient: 4–40% CH 3 CN in 18.34 min. 1 • Column temperature 4 10 13 3 Column temp.: 35 C UV Detection: 220 nm Data Rate: 10 Hz 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 min 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 min Response Time: 0.05 s Method Development Approaches Recommended Method Development Strategies for Method Development Approaches for Peptide Mapping Separations Recommended Method Development Strategies for RPLC-UV and RPLC-MS of Protein Enzymatic Digests for Peptide Mapping Separations Systematic Approach (DryLab) Wang, Carr et al. Approach* Systematic Approach using Computer Simulation (DryLab 4) • Column HALO 2 Peptide ES C18 Systematic Approach (DryLab) 1. Select desired column geometry. 2 Choose initial flow rate based on column ID Wang, Carr et al. Approach 1. Choose desired column geometry. 2 S tfl t f l Computer Simulation (DryLab 4) • Two-dimensional Design – HALO 2 Peptide ES-C18 2.1 x 100 mm, 2 m 2. Choose initial flow rate based on column ID. 3. Select two different gradient times varying by 2- or 3-fold 2. Set flow rate for column max. pressure or 75% of max. pressure. Two dimensional Design – t G x T • Mobile Phase AS l t 0 1% DFA i t by 2- or 3-fold. 4. Select two column temperatures varying by 20–40 C 3. Set final so that last peak of interest elutes at very end of gradient. – 2–3 gradient steepnesses – 2–3 column temperatures – A Solvent: 0.1% DFA in water – B Solvent: 0.1% DFA in CH 3 CN 20–40 C. 5. Set gradient range of 0–5 to 60% CH 3 CN or 0–5 to 50% CH 3 CN 4. Choose temperature based on column stability and “fluidity” 60C? 2 3 column temperatures 3 – initial : 2% B 50% B 30 min 60 min 45 min 0 5 to 50% CH 3 CN. 6. Carry out gradient runs at T1 and T2 with two different gradient times. 5. For example, use 2–50% CH 3 CN for initial run. – final : 50% B • Column Temperatures 60 C 30 min. 60 min. 45 min. two different gradient times. – For example, 30 and 60 min. at 30 and 60 C. 7. Carry out an additional gradient run at 6. Set gradient time for maximum allowable time. Column Temperatures – 30 and 60 C intermediate temperature and gradient time. – For example, 45 min. at 45 C. 8 Et RT d it D Lb ft time. 7. Calculate %B at elution of last peak of interest (using RT %B/min t 0 and t D ) • Gradient Times 30 d 60 i 45 C 8. Enter RTs and areas into DryLab software. – Note: Peak matching can be tricky and difficult 9 Compare the predicted intermediate interest (using RT, %B/min, t 0 , and t D ). 8. Rerun sample with the 2–X %B limit for gradient time corresponding to that final – 30 and 60 min. – 45-min x 50 C run used for 45 C 9. Compare the predicted intermediate gradient run vs. actual intermediate run to assess peak matches. gradient time corresponding to that final %B. peak matching 10. Determine whether there are any optimum regions of robust resolution in the 2-D * Xi li W D i ht St ll Ad S h lli dP t C 30 C resolution map. 11. Optimize linear gradient in gradient editor * Xiaoli Wang, Dwight Stoll, Adam Schellinger, and Peter Carr Peak Capacity Optimization of Peptide Separations in Reversed‐ Phase Gradient Elution Chromatography: Fixed Column Format l h 8 Optimization was carried out to identify a robust gradient time and temperature combination for a run time < 40 minutes or choose a two or three segment gradient to find optimum conditions. 14 Anal. Chem. 2006, 78, 3406‐3416 LC UV Ch t f G di tI tR Summary and Conclusions Peptide Identification Using Proteome Discoverer Software Typical Peptide Mapping Conditions and LC-UV Separations of Enolase Tryptic Digest LC-UV Chromatograms for Gradient Input Runs tT T t F D LbI t Confidence Single Letter Amino Acid Sequence RT [min] Sequest HT Modifications # PSMs # Missed Cleavages Theo. MH+ [Da] XCorr Sequest HT ∆M [ppm] Sequest HT Desirable Mobile Phase Additive Properties Using Different Mobile Phase Additives at Two Temperatures For DryLab Input 30 i di t • A sample of enolase was digested with trypsin, and was analyzed using gradient UHPLC using a 100-mm HALO 2 Peptide ES-C18 High TGAPAR 1.99 1 0 572.31509 1.85 -1.32 High ANIDVK 5.22 1 0 659.37227 1.99 -2.06 High HLADLSK 5 59 2 0 783 43593 2 87 -0 89 10 mM TFA Sample: Enolase Tryptic Digest in 0.5% HCOOH, 1 μg/μL Column: HALO 2 Peptide ES-C18, 2.1 x 100 mm, 2 m Flo 0 3 mL/min 30 min. gradient 60 C 60 min. gradient 60 C using gradient UHPLC using a 100-mm HALO 2 Peptide ES-C18 column. High HLADLSK 5.59 2 0 783.43593 2.87 -0.89 High IATAIEK 6.32 1 0 745.44543 2.26 -1.14 High IGSEVYHNLK 7.76 2 0 1159.6106 2.98 0.04 High DGKYDLDFKNPNSDK 8 46 3 2 1755 81842 5 11 -07 Typical Conditions for RPLC Separations of Peptides Desirable properties for additives Flow: 0.3 mL/min Injection vol.: 1μL Mobile Phase A: 10 mM DFA in water M bil Ph B 10 M DFA i CH CN • Various mobile phase additives were compared in terms of LC-UV d LC MS i l k h k idth d t ti High DGKYDLDFKNPNSDK 8.46 3 2 1755.81842 5.11 -0.7 High LNQLLR 8.75 2 0 756.47265 2.01 1.28 High TFAEALR 8.94 1 0 807.43593 2.06 -1.23 High YDLDFKNPNSDK 9 13 1 1 1455 67505 3 81 0 89 Separations of Peptides • 150 or 250 mm C18 column M bil h A t ith 0 02 additives • Very high purity, quality, stability and reproducibility 18 20 22 Time (min) Mobile Phase B: 10 mM DFA in CH 3 CN Gradient: 2–47% B in 40 min., except as indicated below UV Detection: 220 nm and LC-MS signal, peak shape, peak width and retention. • Difluoroacetic acid (DFA) offers peak shape peak width and High YDLDFKNPNSDK 9.13 1 1 1455.67505 3.81 -0.89 High DGKYDLDFK 9.22 2 1 1100.52587 2.52 -1.5 High KAADALLLK 9.97 2 1 942.59824 2.61 -0.38 High YDLDFK 10 10 1 0 800 3825 1 74 1 28 • Mobile phase A: water with 0.02– 0.2% (v/v) additive and reproducibility • Low UV absorbance with t bl b li d bl k Data Rate: 10 Hz Response Time: 0.05 s • Difluoroacetic acid (DFA) offers peak shape, peak width and retention comparable to TFA and ammonium formate/formic acid, High YDLDFK 10.10 1 0 800.3825 1.74 -1.28 High GNPTVEVELTTEK 10.29 1 0 1416.72167 3.57 -0.73 High VNQIGTLSESIK 10.68 2 0 1288.71071 3.67 -1.3 Hi h SIVPSGASTGVHEALEMR 10 76 1×O id ti [M17] 1 0 1856 91709 4 23 0 29 • Mobile phase B: CH 3 CN with same % additive or 10–30% lower than in acceptable baselines and blank runs but with much better LC-MS ionization efficiency (signal-to-noise). High SIVPSGASTGVHEALEMR 10.76 1×Oxidation [M17] 1 0 1856.91709 4.23 -0.29 High IEEELGDNAVFAGENFHHGDK 11.36 2 0 2328.05273 4.73 -2.91 High AAQDSFAAGWGVMVSHR 11.71 1×Oxidation [M13] 1 0 1805.83878 4.72 -1.41 Hi h SIVPSGASTGVHEALEMR 12 17 2 0 1840 92217 4 22 0 27 A solvent • Temperature: 30 to 60 C • Ability to provide good retention and symmetrical peak shapes for 10 20 30 10 20 10 20 30 40 • Systematic method development for optimizing peptide separations allows one to find temperature and gradient time High SIVPSGASTGVHEALEMR 12.17 2 0 1840.92217 4.22 -0.27 High IEEELGDNAVFAGENFHHGDKL 12.70 1 1 2441.13679 5.34 -0.91 High NVNDVIAPAFVK 13.40 2 0 1286.71031 2.67 -1.51 Temperature: 30 to 60 C • Flow rate set so that linear velocity is 2 mm/sec acidic, basic and zwitterionic analytes Time (min) 10 mM NH 4 COOH + 10 mM HCOOH 10 mM DFA 10 20 Time (min) 10 20 30 40 Time (min) 60 min gradient 30 min. gradient Major peaks were identified in LC-UV chromatograms and entered f separations allows one to find temperature and gradient time combinations for robust, effective analyses. High IGLDCASSEFFK 13.60 1×Carbamidomethyl [C5] 1 0 1373.64058 3.78 -0.46 High AAQDSFAAGWGVMVSHR 13.63 1 0 1789.84386 5.28 -0.45 Hi h TAGIQIVADDLTVTNPK 14 31 2 0 1755 9487 4 97 0 18 is ~2 mm/sec • Initial % CH 3 CN: 5% • Volatile • Acceptably low or no suppression 60 min. gradient 30 C 30 C into Microsoft Excel. Peaks were matched, and then RTs and areas were entered into DryLab 4 software, along with elution conditions. • HALO 2 Peptide ES-C18 columns facilitate high resolution peptide High TAGIQIVADDLTVTNPK 14.31 2 0 1755.9487 4.97 0.18 High LGANAILGVSLAASR 15.02 1 0 1412.82199 4.57 -1.33 High AVDDFLISLDGTANK 15.40 3 0 1578.80098 5.83 -0.48 Hi h WLTGPQLADLYHSLMK 16 89 1 O id ti [M15] 1 0 1888 96258 48 13 • Final % CH 3 CN: 40–60% • Gradient slope: ranges from 0 1% • Acceptably low or no suppression of LC-MS signal Mi ibl ith t d i 16 18 Time (min) 16 18 20 Time (min) For conditions, see panel 8 mapping of complex tryptic digests with short analysis times (20 30 minutes) High WLTGPQLADLYHSLMK 16.89 1×Oxidation [M15] 1 0 1888.96258 4.8 -1.3 High WLTGPQLADLYHSLMK 18.55 2 0 1872.96767 5.32 -0.18 High SGETEDTFIADLVVGLR 19.42 1 0 1821.92288 6.42 -0.33 Gradient slope: ranges from 0.1% to 2% CH 3 CN per min. • Gradient time: can vary from 30 to • Miscible with water and organic modifiers (20–30 minutes). Ak ld t S td i tb NIH G t GM116224 (BEB) High RYPIVSIEDPFAEDDWEAWSHFFK 20.64 1 1 2984.38898 7.39 -1.18 High YPIVSIEDPFAEDDWEAWSHFFK 21.51 2 0 2828.28787 5.46 -0.78 High YGASAGNVGDEGGVAPNIQTAEEALD LIVDAIK 24.66 1 0 3257.61721 4.97 -0.88 • Gradient time: can vary from 30 to 240 min. Note: 2.2–52.2 % B in 40 min. Acknowledgements: Supported in part by NIH Grant GM116224 (BEB). Opinions expressed are solely those of the authors. HALO and Fused- LIVDAIK Data Set was generated using the 30-min, 60 C Xcalibur software raw file PSM = Peptide Spectrum Match XCorr = Cross Correlation Factor 0 10 20 30 Time (min) 0 10 20 30 Time (min) 6 Note: 2.2 52.2 % B in 40 min. 15 Opinions expressed are solely those of the authors. HALO and Fused Core are registered trademarks of Advanced Materials Technology, Inc. Spectrum Match Correlation Factor 12 3 6 DFA, TFA and NH 4 COOH/HCOOH give narrow peaks, similar retention but different selectivities for peptide mapping. 10 20 Time (min) 10 20 30 40 Time (min) 9