Israel lectures 4/10-5/10/2010 ERLIC and Proteomics: Simultaneous Isolation of Phospho- and Glycopeptides and Superior Fractionation of Complex Tryptic Digests Andrew Alpert PolyLC Inc. Columbia, MD U.S.A.
HILIC versus RP: Inverse Selectivity 33,25 100 90 Relative Abundance HILIC PolyLC 80 6,96 26,63 70 19,63 60 Arabidopsis thaliana. leaf extract 50 29,31 40 30 34,03 74,58 61,39 37,69 20 6,24 85,92 41,22 43,65 77,58 8,26 10 22,53 47,81 11,62 17,95 54,04 72,02 62,62 1,25 85,02 0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 Time (min) 64,58 100 RP C18 90 Relative Abundance 80 Hypersil 70 68,42 60 42,39 50 82,23 22,93 40 81,97 61,92 30 22,27 39,47 20 52,12 79,00 21,47 39,05 44,71 83,40 33,95 10 47,64 28,18 2,22 86,27 3,68 12,64 15,64 52,99 72,04 0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 Time (min)
Purification of Variant Glycopeptides from a Tryptic Digest of -Interferon QUESTION: Why can’t you use HILIC to isolate glycopeptides selectively? HILIC of Glycopeptide Asn-97 HILIC of Glycopeptide Asn-25 Adjacent peaks differ by one carbohydrate residue or linkage position HILIC: PolyHYDROXYETHYL A (150x1.0-mm) RPC (Vydac C-18) of total digest from CHO batch culture Data courtesy of J. Zhang and D. Wang (MIT)
ANSWER: Basic residues dominate retention in HILIC 15 = Basic Peptides Column: PolyHYDROXYETHYL A = Acidic Peptides 120 Mobile Phase: 20 mM 16 = Tryptic Peptides Na-MePO 3 , pH 2.0 17 ( = Phosphopeptides ) r.t. (min) 80 14 6 40 1 20 18 19 10 5 13 2,9,12 11 3,4,8 0 45 0 50 55 60 65 70 75 % ACN
Isolation of Tryptic Glycopeptides via HILIC Problem: Basic residues are the most hydrophilic and dominate the chromatography. Addition of a glycan amounts to a small difference between large numbers. Solution: Tune down interaction with basic residues to increase sensitivity to the rest of the peptide. Form hydrophobic Run @ pH 6, not 3 ion pairs with TFA (Steve Carr, 1993) (Wen Ding, 2009) + - CF 3 COO + + ERLIC (Alpert, 2008) - + + + + +
HILIC vs ERLIC Separation of Peptide Standards 1,13 HILIC: PolyHYDROXYETHYL A; 20 mM Na-MePO 3 , pH 2.0, w. 63% ACN 20 ERLIC: PolyWAX LP; 20 mM Na-MePO 3 , pH 2.0, w. 70% ACN = basic peptide = acidic peptide 9,12 4 6 HILIC ABSORBANCE (225 nm) 17 15 Voltage 4 Voltage 1 17 6 20 12 9 13 ERLIC 15 20 0 40 60 80 100 TIME (Min)
ERLIC: Electrostatic Repulsion- Hydrophilic Interaction Chromatography - HILIC on a column of the same charge as the solutes, especially the best-retained solutes -
HILIC
+ + ERLIC + +
ERLIC Mode: Peptide Retention vs. % ACN Column: PolyWAX LP Mobile Phase: 20 mM Na-MePO3, pH 2.0 PROTEOMICS APPLICATION: All peptides elute within a well-defined and adjustable time frame 80 15 16 = Basic Peptides 13 = Acidic Peptides 6 60 = Tryptic Peptides ( = Phosphopeptides) 20 r.t. (min) 40 12 17 20 19 91 11 7 5 4 0 45 0 50 55 60 65 70 75 % ACN
HN HN SOLUTION R R R R HN NH HN NH R = -CH 2 Br, HN NH HN NH Glycidyl, Adsorbed etc. Layer H 2 N NH 2 H 2 N NH 2 - - - - - - - - SURFACE Crosslinking HN HN N N N N HN NH HN NH H 2 N NH 2 H 2 N NH 2 - - - - - - - - - + - + (- X OH ) H 2 O, Salts (X Y ) - Y - Y - Y - Y N H HN + + + N H HN + 3 3 3 3 HN NH HN NH H H N N N N N N + H + + + H H H - - - - - - - - Formation of an Adsorbed Coating of Linear Polyethyleneimine (LPEI)
ERLIC Mode: Peptide Retention vs. pH Column: PolyWAX LP Mobile Phase: 20 mM Na-MePO3 w. 65% ACN 400 = Basic Peptides 9 = Acidic Peptides = Tryptic Peptides 6 300 ( = Phosphopeptides) 13 12 r.t. (min) 11 200 At pH > 3, acidic peptides elute outside the time frame of neutral or basic 100 peptides 20 15 8 7 14 16 18 1,5 0 4 2.0 3.0 4.0 5.0 pH
ERLIC of Synthetic Basic Peptides “ > 95 % pure” Column: PolyWAX LP, 100x4.6-mm Mobile Phase: Na-MePO3 buffer, pH 2.0 - lots of failure sequences & incompletely deprotected side chains; difficult to analyze such peptides any other way - Y R YLQ RRKKK G K A D GGA E YATYQT K STTPA E Q R G 67.5% ACN 70% ACN Y R YLQ RRKKK GTYLT DE T HRE V K FTSL 70% ACN
ERLIC of Nucleotides COLUMN: PolySULFOETHYL A (item# 204SE0503) MOBILE PHASE: 80 mM TEAP, pH 3.0, with 84% ACN; 2 ml/min NADPH 0.04 UTP ATP GTP AMP ABSORBANCE UNITS (260 nm) NADH ADP UMP UDP CTP GMP 0.02 CMP GDP CDP NADP NAD 0.00 40.0 0.0 10.0 30.0 20.0 TIME (Min)
PTM ’ s (Post-Translational Modifications)
CURRENT AFFINITY METHODS FOR VARIOUS PTM’S Phosphopeptides Glycoproteins; Glycopeptides Metal oxides Lectins; antibodies (specific sequences only; (TiO 2 , ZrO 2 , etc.) N-linked, sialylated, etc.) IMAC Metal oxides (TiO2) (sialylated only) [ Larsen et al. ] Hydrazide resin (N-linked only; takes ~ 4 days) ANTI-AFFINITY METHOD SCX (phosphopeptides and sialylated glycopeptides repelled more than other peptides; elute early) [ Gygi et al.; Lewandrowski et al. ]
SELECTIVE ISOLATION OF PHOSPHOPEPTIDES BY ERLIC* EXAMPLE: A tryptic digest at pH 3.0 + A basic residue (N-terminus or C-terminal Arg- or Lys-) A neutral, polar residue - A phosphorylated residue Nonphosphopeptide Phosphopeptide + + + + - PO 4 + + + + + + + + + Phosphate groups promote retention by both electrostatic attraction and hydrophilic interaction. Conditions are selected to make retention of other tryptic peptides marginal. RESULT: Selective isolation of phosphopeptides *Electrostatic Repulsion-Hydrophilic Interaction Chromatography
Tryptic Digest of β -Casein; ERLIC vs. Anion-Exchange 1P : FQ S EEQQQTEDELQDK ar = artifact 4Pa : ELEELNVPGEIVE S L SSS EESITR AP = Alkaline Phosphatase 4Pb : RELEELNVPGEIVE S L SSS EESITR 160 160 ar 4Pb 140 140 4Pa ERLIC 1P ? 120 120 ar 100 100 mVolts mVolts ar 80 80 ANION- 60 60 EXCHANGE ? 40 40 20 20 0 0 0 5 10 15 20 25 30 35 Minutes A.J. Alpert, submitted for publication
ERLIC of HeLa Cell Lysate Tryptic Digest: SPE Desalting of Phosphopeptides Examples of EEDEEGEDVVTSTGR KKEEEEDEEDEEDEEEEEDEEDEDEEEDDDDE sequences from SGGSGGCSGAGGASNCGTGSGR KGDGGGASGGGGGGGGSGGGGSGGGGGGGSSRPPAPQENTTSEAGLPQGEAR SESVV Yp ADIR Sp R Sp Y Tp PEYR these fractions SFTSSSP Sp SPSR KQ Sp FDDND Sp EELEDKDSK Low-salt Fraction High-salt Fraction 25 Artifact 20 Blank run 15 mVolts 10 5 0 A.J. Alpert, 0 5 10 15 20 25 30 35 submitted for Minutes C-18: 133 Phosphopeptides (23%) [1P/2P/3P = 24/79/30], C-18: 102 Phosphopeptides (14%), publication 439 nonphosphopeptides 613 nonphosphopeptides Hyper- 182 Phosphopeptides (49%), Hyper- 52 Phosphopeptides (69%) [1P/2P/3P = 4/27/21], Carb: 189 nonphosphopeptides Carb: 23 nonphosphopeptides
% Phosphopep- 1 1 2 4 5 2 2 16 18 24 26 24 24 20 22 15 53 70 45 34 22 29 10 39 18 92 75 22 12 tide content 400 #Phosphopeptides 3 PO4 300 2 PO4 4 PO4 200 1 PO4 100 0 A280 A D C B BLANK RUN 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Minutes ERLIC of HeLa Cell Lysate Tryptic Digest Sample: 1.5 mg. HeLa digest/200 µl MP A; fractions collected at 1 ’ intervals Column: PolyWAX LP, 100x4.6-mm (104WX0503) DETECTION: 280 nm Flow: 1 ml/min Gradient: A) 20 mM NH4-formate, p H 2.2, w. 70% ACN; B) Same but 10% ACN; C) 1 M NH4-formate, pH 2.2, w. 10% ACN; D) 0.3 M TEAP, pH 2.0, w. 10% ACN The column was eluted with linear gradients of the 4 solvents used for SPE and 29 fractions were collected. These were analyzed via RPC-MS using a LTQ-FT MS [ 4 ]. Over 3000 phosphopeptides were identified with little effort, since the solvent in fractions 1-21 was volatile. This profile serves as a guide to the composition and abundance of phosphopeptides to be expected at various points in the gradients.
Selective Isolation of Tryptic Phosphopeptides on 200- Å PolySULFOETHYL A A A C C AQSGSDS( pS )PEP K 1+ EN( pS )PAAFPD R 1+ 100 100 TVD( pS )P K 1+ TVDSPK 2+ TVDSPK 2+ YFLVGAGAIGCELLK 2+ YFLVGAGAIGCELLK 2+ DS( pS )VPETPDNE R 1+ pH=2.7 pH=2.7 Net charge Net charge LVLDSHIWAFK 3+ LVLDSHIWAFK 3+ LFQLGPP( pS )PV K 1+ OD 220 OD 220 AY( pS )PEY R 1+ NH 3 -Thr- Val-Asp-Ser-Pro-Lys - COOH NH 3 -Thr- Val-Asp-Ser-Pro-Lys - COOH AQSGSDSS*PEPK 1+ AQSGSDSS*PEPK 1+ 2 + 2 + Ac-AEELVLE R 1+ ENS*PAAFPDR 1+ ENS*PAAFPDR 1+ + + + + COOH COOH NH 3 NH 3 TLLEQLDDDQ 1+ TVDS*PK 1+ TVDS*PK 1+ NH 3 -Thr- Val-Asp-Ser-Pro-Lys - COOH NH 3 -Thr- Val-Asp-Ser-Pro-Lys - COOH 1 + 1 + + + + + - - COOH COOH NH 3 NH 3 0 0 HPO 3 HPO 3 0 0 Time (min) Time (min) 35 35 B B D D 100 100 80 80 80 80 XXXXXXS*XXXX(K/R) XXXXXXS*XXXX(K/R) 70 70 70 70 60 60 60 60 % of total % of total OD 220 OD 220 50 50 50 50 40 40 40 40 30 30 30 30 20 20 20 20 10 10 10 10 0 0 0 0 1+ 1+ 2+ 2+ 3+ 3+ 4+ 4+ >4+ >4+ 0 0 0 0 Predicted net charge at pH 2.7 Predicted net charge at pH 2.7 Time (min) Time (min) 35 35 - from Gygi et al. , PNAS 101 (2004) 12130-35 -
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