Can we learn something new about peptide separations after 40 years of RP and HILIC chromatography? Martin Gilar April 12, MASSEP 2016

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Can we learn something new about peptide separations after 40 years of RP and HILIC chromatography? Martin Gilar April 12, MASSEP 2016 2015 Waters Corporation 1

Overview 1. 2D RP RP LC of peptides 2. RP-LC & HILIC separation selectivity (peptides) 3. HILIC for glycopeptides 4. Peptide tailing proline story 5. What are all your peaks in the peptide map? Incomplete digestion Over digestion Protein contaminants 6. Kinetics of tryptic cleavage 2015 Waters Corporation 2

Part 1 2D RP RP LC of peptides 2015 Waters Corporation 3

Orthogonality of LC modes C18, ph 2.6 PFP, ph 3.25 0 50 Retention time (min) 0 60 Retention time (min) C18, ph 10 SEC, ph 4.5 0 50 Retention time (min) 30 45 Retention time (min) HILIC, ph 4.5 SCX, ph 3.25 10 60 Retention time (min) 0 45 Retention time (min) Gilar M. et al., Anal. Chem, 77 (2005) 6426 2015 Waters Corporation 4

Comparing orthogonality 2ndD LC-MS R 2 =? 2nd D time 3 2 5 digest ~200 peptides 1 1 2 3 1st D time 1stD LC-MS 2015 Waters Corporation 5

2D RP RP LC of peptides 1.0 APhenyl x C18 1.0 B PFP x C18 1.0 C18 C ph10 x C18 ph2.6 pi>7.5 pi<5.5 0.8 0.8 0.8 7.5<pI<5.5 RP P henyl, ph 2.6 0.6 0.4 RP P FP, ph 2.6 0.6 0.4 RP C18, ph 10 0.6 0.4 0.2 0.2 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 RP C18, ph 2.6 0.0 0.0 0.2 0.4 0.6 0.8 1.0 RP C18, ph 2.6 0.0 0.0 0.2 0.4 0.6 0.8 1.0 RP C18, ph 2.6 1.0 DSEC x C18 1.0 HILIC E x C18 1+ 1.0 FSCX x C18 1+ 0.8 0.8 2+ 3+ 0.8 2+ 3+ S EC, p H 4.5 0.6 0.4 HILIC, ph 4.5 0.6 0.4 4+ 5+ SC X, ph 3.25 0.6 0.4 4+ 5+ 0.2 0.2 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 RP C18, ph 2.6 0.0 0.0 0.2 0.4 0.6 0.8 1.0 RP C18, ph 2.6 0.0 0.0 0.2 0.4 0.6 0.8 1.0 RP C18, ph 2.6 Gilar M. et al., Anal. Chem, 77 (2005) 6426 2015 Waters Corporation 6

2D RP RP LC of peptides 0-56% B in 70 minutes 20 mm NH4OH ph 10 Bovine_Hemoglobin_Digest_Stored_091803_1 100 28.55 1: Scan ES+ TIC 4.51e9 % 4.29 4.70 6.29 8.91 10.99 11.40 13.24 11.93 14.09 16.30 17.36 18.75 19.61 19.93 22.39 22.79 23.86 26.68 26.51 26.06 27.00 30.68 31.41 35.05 34.27 36.19 ph 2.6 1 Bovine_Hemoglobin_022004_1 100 30.68 1: Scan ES+ TIC 4.37e8 18.95 41.26 ph 10 % 15.79 25.68 29.41 35.85 37.65 5.70 6.77 4.10 5.21 8.53 14.03 10.38 13.21 11.73 9.64 16.41 18.21 18.58 22.39 21.04 19.89 24.20 25.92 29.00 35.48 39.58 41.92 1 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00 Time Gilar M. et al., J. Sep. Sci., 28 (2005) 1694 2015 Waters Corporation 7

Off-line 2D RP RP LC in proteomic Second nanorp LC dimension, ph 2.6 8% peptide overlap between fractions Gilar M. et al., Electrophoresis, 30 (2009) 1157 First RP LC dimension, ph 10 2015 Waters Corporation 8

High-low ph RP RP 2D LC Neue U.D., Mendez A, J. Sep. Sci., 30 (2007) 949 1.0 pi>7.5 1st D LC, C18, ph 10 0.8 0.6 0.4 pi<5.5 7.5<pI<5.5 Bases shift Acids shift 0.2 0.0 0.0 0.2 0.4 0.6 0.8 1.0 2nd D LC, C18 Gilar M. et al., J. Sep. Sci., 28 (2005) 1694 - neutral compounds,,* - tertiary, secondary, multifunctional amines, + - carboxylic, sulfonic acids - phenols. - unclassified analytes 2015 Waters Corporation 9

Part 2 RP LC & HILIC separation selectivity (peptides) 2015 Waters Corporation 10

Retention prediction Peptide retention can be predicted from their AA composition Meek (1980-1981) Guo and Hodges (1986-1987) Wilce and Hearn (1991-1993) Petritis and Smith (2003-2006) Krokhin (2004-2006) Gilar (2007) Proteomic applications R^2 ~ 0.95 0.98 c ln L ( b AA RT b ) 1 i i 0 Gilar M. et al., RCM 21 (2007) 2813 High-low ph prediction Scatter Plot Experimental retention (minutes) 70 60 50 40 30 20 In red: Random decoy sequences 0 10 20 30 40 50 60 Oleg Predicted retention (minutes) 2015 Waters Corporation 11

Retention prediction Could this be used for elucidation of LC condition impact on peptide separation? Ion-pairing effect Separation temperature effect Gradient slope effect 300A vs. 100A columns RP sorbents selectivity 1.0 0.5 c ln L ( b AA RT b ) 1 i i 0 Gilar M. et al., Anal. Chem, 82 (2010) 265 0.0 Ala Asn Cys * Gln Gly Arg Ile Leu Met Phe Pro Ser Thr Trp Tyr Val His Lys Asp -0.5 Glu ph 2.6 ph 10-1.0 Gilar M. et al., RCM 21 (2007) 2813 2015 Waters Corporation 12

Retention prediction workflow RT c ln L ( b AA b ) 1 i i 0 0 ESI MS 5 10 15 20 25 30 35 Fitted retention coefficients of 20 amino acids 1. Collect LC data, training set of peptides of known sequence 2. Establish model, multi-regression fitting of retention coefficients b i 40 Predicted retention 35 30 25 R² = 0.968 HILIC Retention coefficients 6 2 HILIC 20 20 25 30 35 40 Experimental retention 3. Verify prediction success -2 Lys Arg His Glu Asp Gln Asn Cys* Thr Pro Ser Gly Ala Val Tyr Met Ile Leu Phe Trp 4. Retention coefficients b i contain information about LC selectivity 2015 Waters Corporation 13

Impact of ion-pairing 14 12 F W retention coefficients 10 8 6 4 2 G,S,Q,D, T,E,C N A P V,Y M I L 0% TFA (0.1% FA) 0.02% TFA 0.05% TFA 0.1% TFA 0-5 0 5 10 15-2 K R H -4 retention coefficients (0% TFA) Gilar M. et al., Anal. Chem, 82 (2010) 265 2015 Waters Corporation 14

Impact of ion-pairing 2+ 1+ 3+ 4+ 0.1% TFA All 11 AA long 1+ to 4+ charge 4+ 2+ 3+ 1+ 0.05% TFA 4+ 2+ 3+ 1+ 0.02% TFA 3+ 2+ 1+ 4+ 0% TFA (0.1% FA) 16 Minutes 24 Gilar M. et al., Anal. Chem, 82 (2010) 265 2015 Waters Corporation 15

Impact of ion-pairing, 10AA peptides 2 4 1 3 0.1% TFA All 10 AA long All 1+ charge 0.05% TFA 0.02% TFA 2 4 1 3 0% TFA (0.1% FA) 28 Minutes 37 Gilar M. et al., Anal. Chem, 82 (2010) 265 2015 Waters Corporation 16

Impact of temperature retention coefficients 12 10 8 6 4 2 P V Y I L 20 C 30 C 40 C 50 C W 0-2 0 2 4 6 8 10 12-2 K R H retention coefficients (20 C) Gilar M. et al., Anal. Chem, 82 (2010) 265 2015 Waters Corporation 17

PIVL-WY factor Pep Sequence PIVL WY 2 4 1 3 60 C 1 MPIVLGILAG 6 0 2 MWYWYGYYAG 0 6 3 MIPVLGILAT 6 0 4 MYWYWGYYAT 0 6 50 C All 1+ charge All 10 AA long 40 C PIVL-WY factor= (PIVL)- (WY) Gilar M. et al., Anal. Chem, 82 (2010) 265 30 C 2 4 1 3 20 C 28 Minutes 38 2015 Waters Corporation 18

Amide HILIC, ph 4.5 Retention coefficients 8 6 4 2 HILIC retention coefficients 0 Lys Arg His Glu Asp Gln Asn Cys* Thr Pro Ser Gly Ala Val Tyr Met Ile Leu Phe Trp 12 10 8 6 4 2 0-5 0 5 10 15-2 -4-6 -8-10 R² = 0.8373 C18 vs. HILIC (amide) retention coefficient RP C18 retention coefficients -2 Gilar et al., J. Chromatogr. 1218 (2011) 8890 2015 Waters Corporation 19

Impact of silanols, HILIC, ph 4.5 Retention coefficients 16 12 8 4 Silica HILIC BEH HILIC Amide HILIC Tyr Lys His Arg 0-4 0 4 8 12 16 Trp Phe -4 Retention coefficients (Silica HILIC) Gilar et al., J. Chromatogr. 1218 (2011) 8890 2015 Waters Corporation 20

HILIC vs. RP RP LC vs. HILIC Retention coefficients RP LC vs. HILIC Retention time HILIC retention coefficients Lys 16 His Arg 12 8 4 0-4 0 4 8 12 16-4 Silica HILIC Amide HILIC Met Val Ile Leu Phe Trp Tyr RP C18 retention coefficients Silica HILIC HILIC retention retention (minutes) (minutes) 40 55 Silica HILIC 0 basic AA 50 1 basic AA Amide HILIC 2 basic AA 30 45 3 basic AA 4 basic AA 40 20 35 30 10 25 20 0 15 10 20 30 40 0 10 RP LC 20 retention 30(minutes) 40 50 RP LC retention (minutes) Gilar et al., J. Chromatogr. 1218 (2011) 8890 Gilar et al., Anal. Chem. 77 (2005) 6426 2015 Waters Corporation 21

Part 3 HILIC for glycopeptides 2015 Waters Corporation 23

HILIC selective separation of glycopeptides UV 280 nm GLYCOPEPTIDES 4 x magnified IgG tryptic digest 0 5 10 15 20 25 30 35 40 45 minutes MS counts GLYCOPEPTIDES Bovine fetuin Tryptic digest 0 5 10 15 20 25 30 35 40 45 minutes Gilar et al., Anal. Biochem. 417 (2011) 80 2015 Waters Corporation 24

RNAse B glycopeptides 2.1x150 HILIC glycan 0.2ml/min A:10mM ammfa ph 4.5, B:90ACN/10wat tot10mm amfa 90B-55B in4 Man 5 Man 5 oct23_hilic10mm_rnaseh010 2: TOF MS ES+ 100 475.34 0.50Da 520 Man 6 Man 6 UPLC-FL Intact RNAse glycans B % Aglycosylated RNAse Man 7 Man 4 Man 8 Man 7 Man 8 Man 9 Man 9 8 Minutes 14 0 34.00 36.00 38.00 40.00 42.00 44.00 46.00 oct23_hilic10mm_rnaseh010 100 Man 5 NLTK pep 474.28 Da + GlcNac = 678.37 2: TOF MS ES+ 678.37 0.50Da 1.32e3 UPLC-FL glycopeptides % Man 6 Man 4 Man 7 Man 8 Man 9 0 Time 34.00 36.00 38.00 40.00 42.00 44.00 46.00 2015 Waters Corporation 25

UPLC/FLR/MS of N-linked glycans from CD 44 2a 3 non-glycopeptides 12 1) RP-LC fractionation 2) HILIC glycopeptide 1 2b 5 7 11 13 analysis 4 6 8 9 10 12 10 2a 3 1 11 2b 4 5 13 6 7 8 9 Gilar et al., Anal. Biochem. 417 (2011) 80 2015 Waters Corporation 26

Part 4 Peptide tailing proline story 2015 Waters Corporation 27

mab tryptic digest 6.0e-2 1) UPLC-UV (214 nm) at 40 C 5.0e-2 4.0e-2 AU 3.0e-2 2.0e-2 1.0e-2 0.0 7.0e-2 6.0e-2 5.0e-2 2) UPLC-UV (214 nm) at 60 C Proline-rich Peptides AU 4.0e-2 3.0e-2 2.0e-2 1.0e-2 0.0 10 20 30 40 50 60 70 80 RetentionTime (min) 2015 Waters Corporation 28

mab tryptic digest proline-rich peptides Waters application 720003063 2015 Waters Corporation 29

Part 5 What are all those peaks in your peptide map? 2015 Waters Corporation 30

Sample complexity 0.08 Enolase peptide mapping (UPLC) 0.06 AU 0.04 0.02 0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 Minutes Non-specific cleavages, missed cleavages, point mutations, post translational modifications, glycoforms, chemical modifications, protein contaminations 2015 Waters Corporation 31

Enolase LC/MS E analysis Enolase Tryptic Enolase semi-tryptic non-tryptic *Enolase-deamidation Other proteins (Buy one, get one free) Xie et al., Anal. Chem. 81 (2009) 5699 2015 Waters Corporation 32

Part 6 Kinetics of tryptic cleavage 2015 Waters Corporation 33

Tryptic digestion kinetics LYAA[R]LYAVR LYAA[R] + LYAVR [R], [K] [DR], [DK] [ER], [EK] [RD], [KD] [RE], [KE] [RTD], [KTD] [RTE], [KTE] [DTR], [DTK] [ETR], [ETK] [RR], [KK] [RK], [KR] Slechtova et al., Anal. Chem. 87 (2016) 7636 2015 Waters Corporation 34

Proteins identified by LC/MS E analysis LYAA[RR]LYAVR LYAA[RR] + LYAVR or LYAA[R] + [R]LYAVR LYAA[RR] LYAA[R] + R [R]LYAVR LYAVR + R Slechtova et al., Anal. Chem. 87 (2016) 7636 2015 Waters Corporation 35

Some sequences digest 100-1000 x slower 100 90 100.0 [DR], [DK] [DTR], [DTK] [ETR], [ETK] Strong inhibition! Tryptic digestion relative speed (%) 80 70 60 50 40 30 20 10 0 73.0 52.2 48.6 32.3 26.1 45.1 44.5 44.4 36.8 30.2 20.6 21.4 7.7 LYAARR RLYAVR 33.4 5.4 5.8 2.3 11.2 4.5 3.1 7.8 0.3 0.6 0.036 0.006 [R] [K] [RR] [KK] [KR] [RK] [RTE] [KTE] [RE] [KE] [RTD] [KTD] [ER] [EK] [RD] [KD] LYAAR*R LYAAK*K [ETR] [ETK] [DR] [DK] [DTR] [DTK] R*LYAVR K*LYAVR Very slow! Extremely slow! Slechtova et al., Anal. Chem. 87 (2016) 7636 2015 Waters Corporation 36

Thank you! Questions? 2015 Waters Corporation 37

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