RP RP High Efficiency LC/MS Analysis of O GlcNAc Modified Synthetic Peptides High Efficiency LC/MS Analysis of O‐GlcNAc Modified Synthetic Peptides 123 l 3 h i h 1 l d 23 Barry Boyes 1, 2, 3 ; Alex Harvey 3 ; Stephanie Schuster 1 ; Ron Orlando 2, 3 1 Advanced Materials Technology Inc Wilmington DE; 2 Complex Carbohydrate Research Center University of Georgia Athens GA; 3 GlycoScientific Inc Athens GA 1 Advanced Materials Technology Inc., Wilmington, DE; 2 Complex Carbohydrate Research Center, University of Georgia, Athens, GA; 3 GlycoScientific, Inc., Athens, GA f f /G C/ SC f C f /G C /G Objectives Prediction of Resolution of Peptide/Glycopeptide Pairs LC/MS Conditions for RP and HILIC of Peptides/Glycopeptides HILIC Better Resolves Matched Peptide/Glycopeptide Pairs Objectives E i di i f f i hi h l i LC/MS i f • A mix of Ammonium Formate/Formic Acid is an attractive mobile phase compared to Formic Acid alone showing narrow • Examine conditions for performing high resolution LC/MS separations of h df d d l l h ff f To uncover new polypeptide glycosylation sites prediction of RP and HILIC retention effects of site occupancy 500 mV 500 mV A mix of Ammonium Formate/Formic Acid is an attractive mobile phase, compared to Formic Acid alone, showing narrow and symmetrical peaks, improved load tolerance (linear isotherm), and good compatibility with online ESI‐MS detection hexosamine modified peptides, particularly the effects of separation RP HILIC To uncover new polypeptide glycosylation sites, prediction of RP and HILIC retention effects of site occupancy by an O‐linked carbohydrate would be useful. As shown below, the correlation of resolution of GP/P pairs 400 450 .866 8 400 450 and symmetrical peaks, improved load tolerance (linear isotherm), and good compatibility with online ESI MS detection (McCalley,J. Chromatogr. A, 1038 (2004), p. 77; Schuster, Boyes, Wagner, Kirkland, J. Chromatogr.A, 1228 (2012), p. 232, mode (RPC and HILIC) APP695‐GP APP695 GP by an O linked carbohydrate would be useful. As shown below, the correlation of resolution of GP/P pairs using RP and HILIC modes of separation is poor, implying that the mechanisms are very different. 350 400 5 6.108 350 400 Johnson, Boyes, Fields, Kopkin, Orlando, Journal of Biomolecular Techniques: JBT (2013).) • Evaluate the utility of LC with online MS for identification and purity A APP695 GP APP695‐Pep APP695‐Pep APP695‐GP Resolution Correlation 300 0.608 300 21.546 • RP and HILIC conditions can employ the same mobile phases, with reversal of the gradient elution composition: RP l d l ( h l ) analysis of hexosamine modified peptides. A 210 APP695‐Pep Resolution Correlation 200 250 200 250 9.485 increases acetonitrile, HILIC decreases acetonitrile (water is the strong solvent). • Figure 1 presents the TIC chromatograms for a standard peptides separated in RP using the Halo Peptide ES C18 column • Define the retention and selectivity differences between RPC and HILIC y = 0 2402x + 1 2808 15.00 150 200 150 200 1 • Figure 1 presents the TIC chromatograms for a standard peptides separated in RP using the Halo Peptide ES‐C18 column and in HILIC mode using the Halo Penta HILIC column Column efficiencies are very comparable and resolution of this mode of separating hexosamine modified and unmodified peptides y = 0.2402x + 1.2808 R² = 0 1379 100 0.688 100 988 and in HILIC mode using the Halo Penta HILIC column. Column efficiencies are very comparable, and resolution of this mixture is readily achieved by either mode of separation. R = 0.1379 10.00 P 0 50 0.486 0 0.854 0 50 22. mixture is readily achieved by either mode of separation. • Retention (Rt), peak widths (W 1/2 ), resolution (Rs) and signal intensities in UV absorbance and in ESI‐MS total ion currents • Introduction Rs R 0.0 2.5 5.0 7.5 10.0 12.5 min 0 15.0 17.5 20.0 22.5 25.0 27.5 min 0 1/2 were determined for the 26 peptides shown in Table 1, using conditions similar to those shown in Figure 1. The Introduction Recent developments HPLC instruments and column packing materials are permitting 5.00 R 1.15 Inten. (x10,000,000) 687 3 9.0 Inten. (x1,000,000) 788.8 27500000 30000000 TIC(+) 156/687 1.15 Inten. (x10,000,000) 687.3 8.5 Inten. (x1,000,000) 788.8 27500000 30000000 TIC(+) glycopeptides and peptides were injected at known quantities, to allow assessment of ESI‐MS signals, normalized to UV‐ Recent developments HPLC instruments and column packing materials are permitting faster separations, for reversed‐phase (RP) and Hydrophilic Interaction Liquid 0.95 1.00 1.05 1.10 687.3 7.5 8.0 8.5 25000000 89 6.1 0 95 1.00 1.05 1.10 7.0 7.5 8.0 25000000 0/687 derived concentration data. Chromatography (HILIC) analyses of peptides, protein fragments and intact proteins. The Figure 1. RP and HILIC Peptide Separations 0.00 0.80 0.85 0.90 60 6.5 7.0 20000000 22500000 5.907/78 0.80 0.85 0.90 0.95 6.0 6.5 20000000 22500000 19.510 89 recent popularity of sub‐2 μm diameter particles and the new development of small 9 Figure 1. RP and HILIC Peptide Separations 2.1 mm ID x 100 mm, 0.35 mL/min, 40°C, MS: SQ TIC (+ 300‐2000 m/z) @ 0.35/s MS 0.00 5.00 10.00 15.00 20.00 25.00 30.00 0 60 0.65 0.70 0.75 5.0 5.5 6.0 17500000 20000000 0.65 0.70 0.75 4.5 5.0 5.5 17500000 20000000 21.588/7 diameter superficially porous particles (SPP) designed for biomolecules is permitting f t dhi h l ti ti f tid d ti f t 3000000 TIC(+) 1.562/499 /478 7/464 7 S5 S4 S3 S1 HILIC: 100‐10%B in 45 min (‐1% AcN/min) MS Rs HILIC 0.45 0.50 0.55 0.60 3.5 4.0 4.5 15000000 0.45 0.50 0.55 0.60 3.5 4.0 15000000 faster and higher resolution separations of peptides and protein fragments. 2500000 11 12.717/ 13.65 4.287/45 549/443 S5 S4 S2 HILIC: 100‐10%B in 45 min (‐1% AcN/min) B‐90%AcN/0.1% FA/10 mM AmmForm A 40%AcN/0 1% FA/10 mM AmmForm Given that the peak widths are the same for HILIC and RP, improved HILIC resolution of GP/P pairs must be 0.30 0.35 0.40 2.0 2.5 3.0 10000000 12500000 0.30 0.35 0.40 2.0 2.5 3.0 10000000 12500000 Modification of serine or threonine residues of proteins by β‐D‐N‐acetylglucosamine 2000000 14 17.5 A‐40%AcN/0.1% FA/10 mM AmmForm Halo Penta‐HILIC Given that the peak widths are the same for HILIC and RP, improved HILIC resolution of GP/P pairs must be driven by selectivity differences. Comparing the plot of the differences in retention time (ΔRt, representing 0.10 0.15 0.20 0.25 05 1.0 1.5 7500000 10000000 0 10 0.15 0.20 0.25 1.0 1.5 7500000 10000000 Modification of serine or threonine residues of proteins by β D N acetylglucosamine (GlcNAc) has emerged as a significant biological signaling mechanism. O‐GlcNAcylation of 1500000 AcN difference at elution) for each mode, for each peptide pair, also yields a poor correlation. 500 750 1000 1250 1500 1750 m/z 0.00 0.05 1372.9 500 750 1000 1250 1500 1750 m/z 0.0 0.5 1051.5 1577.1 5000000 500 750 1000 1250 1500 1750 m/z 0.00 0.05 0.10 1372.9 636.6 500 750 1000 1250 1500 1750 m/z 0.0 0.5 687.3 1577.1 1051.6 5000000 310/723 relevant sites can involve meaningful cross‐talk with phosphorylation targets, both 1000000 h l i f l i b h i bili f i f i d 0 2500000 0 2500000 20.3 nearby and at distant sequences. Similarly modification of polypeptides by α‐GalNAc 500000 MUC5AC3 The poor correlation of ΔRt results, in part, by the greater variability of separation performance in RP mode, and the smaller effect of the HexNAc modification on retention for RP the mean value of ΔRt (Table 1) is 700 mV 800 mV 52 0.0 2.5 5.0 7.5 10.0 12.5 min 0 15.0 17.5 20.0 22.5 25.0 27.5 min 0 (mucin antigen Tn) is of significant interest. For a variety of purposes, we have prepared 8 30 id h i id ih d ih O Gl NA difi i i d 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min TIC(+) 3000000 RP HILIC MUC5AC3/13 MUC5AC13 MUC5AC3 and the smaller effect of the HexNAc modification on retention; for RP the mean value of ΔRt (Table 1) is ‐0 73 (74% rsd) compared with 2 17 (22% rsd) for HILIC HILIC is better in both absolute terms for resolving 600 9 20.465 700 7.75 8.444 .304 8‐30 residue synthetic peptides with and without O‐GlcNAc modifications at serine and threonine residues LC/MS methods to qualify the purity and identities of such peptides 2500000 S5 S4 S3 RP:2‐58.2%B in B in 45 min (1% AcN/min) B 80%A N/0 1% FA/10 M A F RP HILIC MUC5AC MUC5AC3/13 MUC5AC MUC5AC3/13 ‐0.73 (74% rsd), compared with 2.17 (22% rsd) for HILIC. HILIC is better in both absolute terms for resolving GP/P pairs, as well as less variable in the retention effect of a HexNAc addition to the peptide sequences. 500 600 16.406 496 18.669 2 600 8.523 9. threonine residues. LC/MS methods to qualify the purity and identities of such peptides using both Hydrophilic‐interaction chromatography (HILIC) and reversed‐phase 2000000 91/464 411/478 333/499 S2 B‐80%AcN/0.1% FA/10 mM AmmForm A‐0.1% FA/10 mM AmmForm l id S 8 A MUC5AC MUC5AC3 MUC5AC GP/P pairs, as well as less variable in the retention effect of a HexNAc addition to the peptide sequences. Δ Rt Correlation 400 500 18.4 500 using both Hydrophilic‐interaction chromatography (HILIC) and reversed‐phase chromatography (RPC) are compared. With many peptide/glycopeptide pairs we observe 1500000 2000000 35/443 805/457 10.79 13.4 16.3 S1 Halo Peptide ES‐C18 A 210 MUC5AC3 MUC5AC13 Δ Rt Correlation 400 400 chromatography (RPC) are compared. With many peptide/glycopeptide pairs we observe much improved separations and LC/MS features using HILIC, compared to RPC. LC/MS 1500000 8.03 9. S1 0 6387 0 6484 4 300 8 300 0.610 using high performance HILIC permits rapid and sensitive analysis of O‐GlcNAcylated 1000000 y = 0.6387x ‐ 0.6484 R² = 0 3266 3 200 88 88 70 16.878 8 6 039 0 200 862 .275 8 8.845 peptides. A collection of sequence‐matched glycopeptide/peptide pairs are compared for 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min 500000 R = 0.3266 RP 100 15.58 15.88 16.77 17.31 17.75 19. 19.41 22.430 100 96 0.690 0.8 34 7.520 8.183 8. 8.708 9.056 9.565 various separation features in RP and HILIC mode, as well as for detection by absorbance tl l th (210 ) d li l t i i ti t t (ESI Table 1 Paired comparison of RP and HILIC for Glycopeptide/Peptide LC/MS Th 26 tid h l db LC/MS i 2 Δ Rt 10.0 12.5 15.0 17.5 20.0 22.5 min 0 0.0 2.5 5.0 7.5 10.0 12.5 min 0 0.49 1.13 7 9 at low wavelength (210 nm) and on‐line electrospray ionization mass spectrometry (ESI‐ MS) using the single quadrupole analyzer Table 1. Paired comparison of RP and HILIC for Glycopeptide/Peptide LC/MS. The 26 peptides shown were analyzed by LC/MS using 2.1 x 100 mm columns, with a flow rate of 0.4 mL/min at 60°C. Gradient conditions: A – 0.1% formic acid/10 mM ammonium formate; B – 90% 1 Δ 3 75 4.00 Inten. (x1,000,000) 954.9 5.25 5.50 Inten. (x1,000,000) 751.8 14000000 15000000 TIC(+) 14000000 15000000 TIC(+) 55 MS) using the single quadrupole analyzer. 2.1 x 100 mm columns, with a flow rate of 0.4 mL/min at 60 C. Gradient conditions: A 0.1% formic acid/10 mM ammonium formate; B 90% Acetonitrile in A. 500 pmol was injected, with detection at 210 nm, MS operated +4.5 kV, with 0.45 s scan from 500 to 2000 m/z. RP Gradient ‐ 4% to / ( / ) / ( / ) f 1 0 95 1.00 1.05 Inten. (x1,000,000) 751.7 3.25 3.50 3.75 4.50 4.75 5.00 13000000 14000000 92/955 13000000 14000000 7.792/95 339/752 Materials and Methods 34% AcN/30 min (1%/min); HILIC Gradient ‐ 90% to 60% AcN/30 min (‐1%/min). The sequences were selected based on known O‐HexNAc modified sites in a variety of proteins Peak widths; means were 0 092 min for RP and 0 090 min for HILIC Average and range of Rt for the modes were similar 0 0.80 0.85 0.90 0.95 2.75 3.00 3.50 3.75 4.00 4.25 11000000 12000000 20.49 11000000 12000000 9.3 Materials and Methods Cl f HALO P tid ES C18 dH l P t HILIC d d t Ad d in a variety of proteins. Peak widths; means were 0.092 min. for RP and 0.090 min. for HILIC. Average and range of Rt for the modes were similar. M Δ R RP Δ R HILIC 0.00 1.00 2.00 3.00 4.00 0.65 0.70 0.75 1502.1 2.00 2.25 2.50 2.75 3.00 3.25 9000000 10000000 752 9000000 10000000 5/853 7 Columns of HALO Peptide ES‐C18 and Halo Penta HILIC were produced at Advanced Materials Technology Inc (Wilmington DE) Both materials employ Fused Core® silica Peptide Description Sequence Mass (neutral) Rt RP (min) Δ Rt RP (GP‐P) Rs RP Rt HILIC (min) Δ Rt HILIC (GP‐P) Rs HILIC APP695‐14GPep VPTT(OGlcNAc)AASTPDAVDK 1574 8 5 87 21 55 MS Δ Rt HILIC 0.50 0.55 0.60 1.50 1.75 2.00 2.25 2.50 8000000 9000000 16.465/7 6 /853 8000000 9000000 8.475 550/1577 Conclusions and Future Directions Materials Technology Inc. (Wilmington, DE). Both materials employ Fused‐Core silica particles of 2.7 μm diameter, a solid core of 1.7 μm diameter, and a shell thickness of 0.5 APP695 14GPep VPTT(OGlcNAc)AASTPDAVDK 1574.8 5.87 21.55 APP695‐14Pep VPTTAASTPDAVDK 1371.7 6.11 ‐0.24 1.90 19.49 2.07 9.41 MUC5AC GTTPSPVPTTSTTSAP 1501.6 9.28 16.41 0.35 0.40 0.45 0 75 1.00 1.25 1 00 1.25 1.50 1.75 6000000 7000000 541/1706 18.700/ 6000000 7000000 8.5 Conclusions and Future Directions particles of 2.7 μm diameter, a solid core of 1.7 μm diameter, and a shell thickness of 0.5 μm. The Halo Peptide shell has 160 Å pores, and the Penta HILIC column 90 Å pores. MUC5AC‐3 GTT(OGalNAc)PSPVPTTSTTSAP 1704.6 8.45 ‐0.83 6.88 18.68 2.27 13.40 MUC5AC‐13 GTTPSPVPTTSTT(OGalNAc)SAP 1704.6 8.53 ‐0.75 5.82 18.51 2.10 10.72 0.20 0.25 0.30 0.25 0.50 0.75 0.25 0.50 0.75 1.00 1502.2 4000000 5000000 18.5 88/904 7 7 4000000 5000000 Synthetic peptides were synthesized at the CCRC (Lab of Prof. Geert‐Jan Boons), or •High performance HILIC and RP HPLC separations were conducted to define preferred mode of MUC5AC3/13 GTT(OGalNAc)PSPVPTTSTT(OGalNAc)SAP 1908.1 7.76 ‐1.52/2 11.84 20.48 4.07/2 23.35 GP‐41 Ac‐CSTFRPRT(OGlcNAc)SSNAST 1758.8 7.09 18.59 0.05 0.10 0.15 694 0 500 750 1000 1250 1500 1750 m/z 0.00 974.0 853.4 1273.1 1909.4 500 750 1000 1250 1500 1750 m/z 0.00 694.2 1007.9 3000000 4000000 19.08 9.467/897 486/1107 3000000 4000000 obtained from AnaSpec (Freemont, CA), or from Colin Mant and Prof. R.S. Hodges (U. High performance HILIC and RP HPLC separations were conducted to define preferred mode of practice to resolve O‐GlcNAc and O‐GalNAc modified peptides. P‐42 Ac‐CSTFRPRTSSNAST 1555.7 7.03 0.06 0.44 17.03 1.56 11.58 GP‐78 Ac‐CQHPPVT(OGlcNAc)NGDTVK 1639.8 6.47 20.32 500 750 1000 1250 1500 1750 m/z 0.00 694.0 1000000 2000000 19 22.4 1000000 2000000 Colorado, Aurora, CO). The Retention Standard Mix uses the S1‐S5 sequences: S1 RGAGGLGLGK A •Formic acid/Ammonium formate mixtures with acetonitrile gradient elution was effective for LC/MS P‐84 Ac‐CQHPPVTNGDTVK 1436.7 6.56 ‐0.10 0.66 18.72 1.61 11.23 GP‐79 Ac‐CKIADFGLS(OGlcNAc)KIVEHQ 1932.0 19.36 19.15 10.0 12.5 15.0 17.5 20.0 22.5 min 0 0.0 2.5 5.0 7.5 10.0 12.5 min 0 S1 RGAGGLGLGK‐Am S2 Ac RGGGGLGLGK Am analysis of the glycopeptide and peptide mixtures using both RP and HILIC operation. Comparing RP versus HILIC for LC/MS analyses of the collection of paired glycopeptides and peptides (GP and P) reveals: P‐85 Ac‐CKIADFGLSKIVEHQ 1728.9 20.80 ‐1.44 8.16 17.21 1.94 14.76 GP‐17s CTLHTKAS(OGlcNAc)GMALLHQ 1854.9 13.62 17.29 S2 Ac‐RGGGGLGLGK‐Am S3 Ac‐RGAGGLGLGK‐Am •HILIC better resolved glycopeptide/peptide mixtures. Comparing RP versus HILIC for LC/MS analyses of the collection of paired glycopeptides and peptides (GP and P) reveals: • As previous observed for unmodified peptides and tryptic digests, RP and HILIC are orthogonal (retention in each mode does not P‐20s CTLHTKASGMALLHQ 1651.8 14.23 ‐0.61 3.06 15.15 2.14 15.38 GP‐15 Ac‐CFELLPT(O‐GlcNAc)PPLSP 1557.8 25.16 5.64 P 18 A CFELLPTPPLSP 1354 7 27 16 2 00 8 88 2 71 2 93 20 11 S3 Ac RGAGGLGLGK Am S4 Ac‐RGVGGLGLGK‐Am •HILIC exhibited some potential to resolve O‐GalNAc modifications at variant sites (3 and 13) on the As previous observed for unmodified peptides and tryptic digests, RP and HILIC are orthogonal (retention in each mode does not significantly correlate to the other). P‐18 Ac‐CFELLPTPPLSP 1354.7 27.16 ‐2.00 8.88 2.71 2.93 20.11 GP‐46 Ac‐CRSSHYGGS(OGlcNAc)LPNVNQI 1975.9 12.48 17.32 P 47 Ac CRSSHYGGSLPNVNQI 1772 8 12 96 0 48 3 83 15 43 1 89 13 91 S5 Ac‐RGVVGLGLGK‐Am MUC5AC peptide sequence. • In all cases, glycopeptides were retained less in RP, eluting at or before the unmodified peptide with the same sequence. P‐47 Ac‐CRSSHYGGSLPNVNQI 1772.8 12.96 ‐0.48 3.83 15.43 1.89 13.91 GP‐51 Ac‐CSALNRTS(OGlcNAc)SDSALHT 1806.8 9.08 17.23 P‐52 Ac‐CSALNRTSSDSALHT 1603 7 9 55 ‐0 47 3 85 15 55 1 69 12 42 The instrument was the Shimadzu Nexera LC‐30 components (40 μL mixer), with the •Additional glycopeptide/peptide pairs will be examined to improve the predictability of retention • In HILIC operation, glycopeptides were retained more, eluting after the unmodified peptide. P‐52 Ac‐CSALNRTSSDSALHT 1603.7 9.55 ‐0.47 3.85 15.55 1.69 12.42 GP‐16 Ac‐CKIPGVS(OGlcNAc)TPQTL 1487.7 16.41 13.27 P‐19 Ac‐CKIPGVSTPQTL 1284.6 16.98 ‐0.58 3.74 10.59 2.68 21.63 SPD20 Absorbance detector, fitted with a high sensitivity semi‐micro flow cell, and the MS 2020 i l d l MS d 4 5 kV ill il i prediction for O‐HexNAc peptide modifications. • The difference in retention time (%AcN) between paired GP and P were much larger in HILIC than RP. F ll i f l tid / tid l ti i hi h i HILIC th RP P 19 Ac CKIPGVSTPQTL 1284.6 16.98 0.58 3.74 10.59 2.68 21.63 GP‐2‐p53 Ac‐CQLWVDS(OGlcNAc)TPPPG 1543.7 16.43 12.72 P‐3‐p53 Ac‐CQLWVDSTPPPG 1340.6 17.66 ‐1.23 7.23 10.41 2.31 10.28 MS‐2020 single quadrupole MS, operated at +4.5 kV capillary potential, scanning between 300 2000 m/z at 0 3 or 0 45 sec/scan Chromatographic parameter measures d b • For all pairs of glycopeptides/peptides, resolution is higher in HILIC than RP. • TIC signal is slightly lower ( 35%) in RP than HILIC for either GP or P but variable with sequence This was the case comparing either GP‐17r Ac‐CLHTKAS(OGlcNAc)GMALL 1488.7 16.21 10.59 P‐20r Ac‐CLHTKASGMALL 1285.6 16.98 ‐0.77 2.79 7.45 3.14 24.73 between 300‐2000 m/z at 0.3 or 0.45 sec/scan. Chromatographic parameter measures used the absorbance data at 210 nm generated with LabSolutions v 5 54 software •Supported by NIH NIGMS GM099355, GM093747. • TIC signal is slightly lower (‐35%) in RP than HILIC, for either GP or P, but variable with sequence. This was the case comparing either all peptides and glycopeptides as groups or by pair‐wise GP/P comparisons HILIC conditions appear less noisy Average 13.01 ‐0.73 4.93 15.29 2.17 15.21 used the absorbance data at 210 nm, generated with LabSolutions v.5.54 software.. all peptides and glycopeptides as groups, or by pair wise GP/P comparisons. HILIC conditions appear less noisy. Standard Deviation 5.95 0.54 3.32 4.74 0.47 5.13 % RSD 45.7 74.3 67.3 31.0 21.8 33.7