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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1 Can we learn something new about peptide separations after 40 years of RP and HILIC chromatography? Martin Gilar April 12, MASSEP Waters Corporation 1
2 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
3 Part 1 2D RP RP LC of peptides 2015 Waters Corporation 3
4 Orthogonality of LC modes C18, ph 2.6 PFP, ph Retention time (min) 0 60 Retention time (min) C18, ph 10 SEC, ph Retention time (min) Retention time (min) HILIC, ph 4.5 SCX, ph Retention time (min) 0 45 Retention time (min) Gilar M. et al., Anal. Chem, 77 (2005) Waters Corporation 4
5 Comparing orthogonality 2ndD LC-MS R 2 =? 2nd D time digest ~200 peptides st D time 1stD LC-MS 2015 Waters Corporation 5
6 2D RP RP LC of peptides 1.0 APhenyl x C B PFP x C C18 C ph10 x C18 ph2.6 pi>7.5 pi< <pI<5.5 RP P henyl, ph RP P FP, ph RP C18, ph RP C18, ph RP C18, ph RP C18, ph DSEC x C HILIC E x C FSCX x C S EC, p H HILIC, ph SC X, ph RP C18, ph RP C18, ph RP C18, ph 2.6 Gilar M. et al., Anal. Chem, 77 (2005) Waters Corporation 6
7 2D RP RP LC of peptides 0-56% B in 70 minutes 20 mm NH4OH ph 10 Bovine_Hemoglobin_Digest_Stored_091803_ : Scan ES+ TIC 4.51e9 % ph Bovine_Hemoglobin_022004_ : Scan ES+ TIC 4.37e ph 10 % Time Gilar M. et al., J. Sep. Sci., 28 (2005) Waters Corporation 7
8 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 Waters Corporation 8
9 High-low ph RP RP 2D LC Neue U.D., Mendez A, J. Sep. Sci., 30 (2007) pi>7.5 1st D LC, C18, ph pi< <pI<5.5 Bases shift Acids shift nd D LC, C18 Gilar M. et al., J. Sep. Sci., 28 (2005) neutral compounds,,* - tertiary, secondary, multifunctional amines, + - carboxylic, sulfonic acids - phenols. - unclassified analytes 2015 Waters Corporation 9
10 Part 2 RP LC & HILIC separation selectivity (peptides) 2015 Waters Corporation 10
11 Retention prediction Peptide retention can be predicted from their AA composition Meek ( ) Guo and Hodges ( ) Wilce and Hearn ( ) Petritis and Smith ( ) Krokhin ( ) Gilar (2007) Proteomic applications R^2 ~ 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) In red: Random decoy sequences Oleg Predicted retention (minutes) 2015 Waters Corporation 11
12 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 c ln L ( b AA RT b ) 1 i i 0 Gilar M. et al., Anal. Chem, 82 (2010) 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 Gilar M. et al., RCM 21 (2007) Waters Corporation 12
13 Retention prediction workflow RT c ln L ( b AA b ) 1 i i 0 0 ESI MS 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 R² = HILIC Retention coefficients 6 2 HILIC 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
14 Impact of ion-pairing F W retention coefficients 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 K R H -4 retention coefficients (0% TFA) Gilar M. et al., Anal. Chem, 82 (2010) Waters Corporation 14
15 Impact of ion-pairing % TFA All 11 AA long 1+ to 4+ charge % TFA % TFA % TFA (0.1% FA) 16 Minutes 24 Gilar M. et al., Anal. Chem, 82 (2010) Waters Corporation 15
16 Impact of ion-pairing, 10AA peptides % TFA All 10 AA long All 1+ charge 0.05% TFA 0.02% TFA % TFA (0.1% FA) 28 Minutes 37 Gilar M. et al., Anal. Chem, 82 (2010) Waters Corporation 16
17 Impact of temperature retention coefficients P V Y I L 20 C 30 C 40 C 50 C W K R H retention coefficients (20 C) Gilar M. et al., Anal. Chem, 82 (2010) Waters Corporation 17
18 PIVL-WY factor Pep Sequence PIVL WY C 1 MPIVLGILAG MWYWYGYYAG MIPVLGILAT MYWYWGYYAT C All 1+ charge All 10 AA long 40 C PIVL-WY factor= (PIVL)- (WY) Gilar M. et al., Anal. Chem, 82 (2010) C C 28 Minutes Waters Corporation 18
19 Amide HILIC, ph 4.5 Retention coefficients HILIC retention coefficients 0 Lys Arg His Glu Asp Gln Asn Cys* Thr Pro Ser Gly Ala Val Tyr Met Ile Leu Phe Trp R² = C18 vs. HILIC (amide) retention coefficient RP C18 retention coefficients -2 Gilar et al., J. Chromatogr (2011) Waters Corporation 19
20 Impact of silanols, HILIC, ph 4.5 Retention coefficients Silica HILIC BEH HILIC Amide HILIC Tyr Lys His Arg Trp Phe -4 Retention coefficients (Silica HILIC) Gilar et al., J. Chromatogr (2011) Waters Corporation 20
21 HILIC vs. RP RP LC vs. HILIC Retention coefficients RP LC vs. HILIC Retention time HILIC retention coefficients Lys 16 His Arg Silica HILIC Amide HILIC Met Val Ile Leu Phe Trp Tyr RP C18 retention coefficients Silica HILIC HILIC retention retention (minutes) (minutes) Silica HILIC 0 basic AA 50 1 basic AA Amide HILIC 2 basic AA basic AA 4 basic AA RP LC 20 retention 30(minutes) RP LC retention (minutes) Gilar et al., J. Chromatogr (2011) 8890 Gilar et al., Anal. Chem. 77 (2005) Waters Corporation 21
22 Part 3 HILIC for glycopeptides 2015 Waters Corporation 23
23 HILIC selective separation of glycopeptides UV 280 nm GLYCOPEPTIDES 4 x magnified IgG tryptic digest minutes MS counts GLYCOPEPTIDES Bovine fetuin Tryptic digest minutes Gilar et al., Anal. Biochem. 417 (2011) Waters Corporation 24
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 Da 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 oct23_hilic10mm_rnaseh Man 5 NLTK pep Da + GlcNac = : TOF MS ES Da 1.32e3 UPLC-FL glycopeptides % Man 6 Man 4 Man 7 Man 8 Man 9 0 Time Waters Corporation 25
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 analysis a b Gilar et al., Anal. Biochem. 417 (2011) Waters Corporation 26
26 Part 4 Peptide tailing proline story 2015 Waters Corporation 27
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 e-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 RetentionTime (min) 2015 Waters Corporation 28
28 mab tryptic digest proline-rich peptides Waters application Waters Corporation 29
29 Part 5 What are all those peaks in your peptide map? 2015 Waters Corporation 30
30 Sample complexity 0.08 Enolase peptide mapping (UPLC) 0.06 AU Minutes Non-specific cleavages, missed cleavages, point mutations, post translational modifications, glycoforms, chemical modifications, protein contaminations 2015 Waters Corporation 31
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) Waters Corporation 32
32 Part 6 Kinetics of tryptic cleavage 2015 Waters Corporation 33
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) Waters Corporation 34
![[R]LYAVR LYAA[RR] LYAA[R] + R [R]LYAVR LYAVR + R](/docs-images/89/97979071/images/34-3.jpg "Slechtova et al., Anal. Chem.")
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) Waters Corporation 35
35 Some sequences digest x slower [DR], [DK] [DTR], [DTK] [ETR], [ETK] Strong inhibition! Tryptic digestion relative speed (%) LYAARR RLYAVR [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) Waters Corporation 36
36 Thank you! Questions? 2015 Waters Corporation 37
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