Complex Tryptic Digests Andrew Alpert PolyLC Inc. Columbia, MD - - PowerPoint PPT Presentation

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

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 Time (min) 10 20 30 40 50 60 70 80 90 100 Relative Abundance 33,25 6,96 26,63 19,63 29,31 34,03 74,58 61,39 37,69 6,24 85,92 41,22 43,65 77,58 8,26 22,53 47,81 54,04 17,95 72,02 11,62 62,62 85,02 1,25 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 Time (min) 10 20 30 40 50 60 70 80 90 100 Relative Abundance 64,58 68,42 42,39 82,23 22,93 81,97 61,92 22,27 39,47 52,12 79,00 44,71 39,05 21,47 83,40 33,95 47,64 28,18 2,22 86,27 15,64 52,99 3,68 12,64 72,04

HILIC PolyLC RP C18 Hypersil

Arabidopsis thaliana. leaf extract

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 HILIC: PolyHYDROXYETHYL A (150x1.0-mm) RPC (Vydac C-18) of total digest from CHO batch culture

Adjacent peaks differ by one carbohydrate residue or linkage position

Data courtesy of J. Zhang and D. Wang (MIT)

ANSWER: Basic residues dominate retention in HILIC

40 80 120 45 50 55 60 65 70 75

% ACN r.t. (min)

= Basic Peptides = Acidic Peptides = Tryptic Peptides ( = Phosphopeptides)

15 16 17 14 6 1 20 18 19 10 13 5 2,9,12 3,4,8 11

Column: PolyHYDROXYETHYL A Mobile Phase: 20 mM Na-MePO3, pH 2.0

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.

+ +

Run @ pH 6, not 3 (Steve Carr, 1993)

+-

CF3COO

Form hydrophobic ion pairs with TFA (Wen Ding, 2009) Solution: Tune down interaction with basic residues to increase sensitivity to the rest of the peptide.

• ERLIC (Alpert, 2008)

+ + + + +

HILIC vs ERLIC Separation of Peptide Standards

HILIC: PolyHYDROXYETHYL A; 20 mM Na-MePO3, pH 2.0, w. 63% ACN ERLIC: PolyWAX LP; 20 mM Na-MePO3, pH 2.0, w. 70% ACN

TIME (Min)

Voltage

Voltage HILIC

15 17 6 20 1,13 9,12 4 4

ERLIC

13 15 6 20 12 9 17 1

20 40 60 80 100

ABSORBANCE (225 nm)

= basic peptide = acidic peptide

ERLIC:

Electrostatic Repulsion- Hydrophilic Interaction Chromatography

• HILIC on a column of the same charge as the

solutes, especially the best-retained solutes -

HILIC

ERLIC

+ + + +

20 40 60 80 45 50 55 60 65 70 75 % ACN r.t. (min)

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

= Basic Peptides = Acidic Peptides = Tryptic Peptides ( = Phosphopeptides)

15 16 13 6 20 12 17 19 91 11 7 5 4

R R R R HN HN NH HN NH H2N NH2 HN HN NH HN NH H2N NH2 HN N N HN NH H2N NH2 HN N N HN NH H2N NH2 N N N HN NH N H HN H

3 3

N N N HN NH N H HN H

3 3

+ +

• Y
• Y

+ +

• Y
• Y

+

H

+

H

+

H

+

H Crosslinking H2O, Salts (X Y )

+ -

• (- X OH )

+

• SOLUTION

Adsorbed Layer SURFACE R = -CH2Br, Glycidyl, etc.

Formation of an Adsorbed Coating of Linear Polyethyleneimine (LPEI)

100 200 300 400 2.0 3.0 4.0 5.0

pH r.t. (min)

= Basic Peptides = Acidic Peptides = Tryptic Peptides ( = Phosphopeptides)

ERLIC Mode: Peptide Retention vs. pH

Column: PolyWAX LP Mobile Phase: 20 mM Na-MePO3 w. 65% ACN

11 20 8 7 18 1,5 4 9 6 13 12 15 14 16

At pH > 3,

acidic peptides elute outside the time frame of neutral or basic peptides

YRYLQRRKKKGKADGGAEYATYQTKSTTPAEQRG 67.5% ACN 70% ACN YRYLQRRKKKGTYLTDETHREVKFTSL 70% ACN

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 -

ERLIC of Nucleotides

COLUMN: PolySULFOETHYL A (item# 204SE0503) MOBILE PHASE: 80 mM TEAP, pH 3.0, with 84% ACN; 2 ml/min

TIME (Min)

0.0 10.0 20.0 30.0 40.0

ABSORBANCE UNITS (260 nm)

0.00 0.02 0.04

CTP NADP NAD GTP ATP UTP NADPH CDP CMP GDP GMP ADP AMP NADH UMP UDP

PTM’s

(Post-Translational Modifications)

CURRENT AFFINITY METHODS FOR VARIOUS PTM’S

Phosphopeptides Glycoproteins; Glycopeptides Metal oxides Lectins; antibodies (specific sequences only; (TiO2, ZrO2, 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

+ +

+ + + + + + + + +

+ +

PO4

• Phosphate groups promote retention by both electrostatic attraction and

hydrophilic interaction. Conditions are selected to make retention of

• ther tryptic peptides marginal.

RESULT: Selective isolation of phosphopeptides *Electrostatic Repulsion-Hydrophilic Interaction Chromatography

Nonphosphopeptide Phosphopeptide

5 10 15 20 25 30 35

20 40 60 80 100 120 140 160

20 40 60 80 100 120 140 160

Tryptic Digest of β-Casein; ERLIC vs. Anion-Exchange

1P: FQSEEQQQTEDELQDK ar = artifact 4Pa: ELEELNVPGEIVESLSSSEESITR AP = Alkaline Phosphatase 4Pb: RELEELNVPGEIVESLSSSEESITR ERLIC ANION- EXCHANGE

1P 4Pa

? ?

ar ar ar 4Pb

A.J. Alpert, submitted for publication

Minutes 5 10 15 20 25 30 35 mVolts 5 10 15 20 25

Low-salt Fraction High-salt Fraction Blank run Artifact

ERLIC of HeLa Cell Lysate Tryptic Digest: SPE Desalting of Phosphopeptides

C-18: 102 Phosphopeptides (14%), 613 nonphosphopeptides Hyper- 182 Phosphopeptides (49%), Carb: 189 nonphosphopeptides C-18: 133 Phosphopeptides (23%) [1P/2P/3P = 24/79/30], 439 nonphosphopeptides Hyper- 52 Phosphopeptides (69%) [1P/2P/3P = 4/27/21], Carb: 23 nonphosphopeptides

KKEEEEDEEDEEDEEEEEDEEDEDEEEDDDDE KGDGGGASGGGGGGGGSGGGGSGGGGGGGSSRPPAPQENTTSEAGLPQGEAR SpRSpYTpPEYR KQSpFDDNDSpEELEDKDSK EEDEEGEDVVTSTGR SGGSGGCSGAGGASNCGTGSGR SESVVYpADIR SFTSSSPSpSPSR

Examples of sequences from these fractions

A.J. Alpert, submitted for publication

100 200 300 400

Minutes

2 4 6 8 10 12 14 16 18 20 22 24 26 28

A280

BLANK RUN

#Phosphopeptides

1 1 2 4 5 2 2 16 18 24 26 24 20 24 22 15 53 45 70 34 22 10 29 39 18 75 92 22 12

% Phosphopep- tide content

1 PO4 2 PO4 3 PO4 4 PO4 A B C D 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.

10 20 30 40 50 60 70 80 10 20 30 40 50 60 70 80

% of total Predicted net charge at pH 2.7 1+ 2+ 3+ 4+ >4+

NH3-Thr- Val-Asp-Ser-Pro-Lys -COOH NH3 COOH

pH=2.7 Net charge 2+ 1+

HPO3

• +

+

A B C

NH3-Thr- Val-Asp-Ser-Pro-Lys -COOH NH3 COOH

+ +

Time (min) 100 35 OD220 Time (min) OD220 100 35

AQSGSDSS*PEPK 1+ ENS*PAAFPDR 1+ TVDS*PK 1+ TVDSPK 2+ YFLVGAGAIGCELLK 2+ LVLDSHIWAFK 3+

D

XXXXXXS*XXXX(K/R) 10 20 30 40 50 60 70 80 10 20 30 40 50 60 70 80

% of total Predicted net charge at pH 2.7 1+ 2+ 3+ 4+ >4+

NH3-Thr- Val-Asp-Ser-Pro-Lys -COOH NH3 COOH

pH=2.7 Net charge 2+ 1+

HPO3

• +

+

A B C

NH3-Thr- Val-Asp-Ser-Pro-Lys -COOH NH3 COOH

+ +

Time (min) 100 35 OD220 Time (min) OD220 100 35

AQSGSDSS*PEPK 1+ ENS*PAAFPDR 1+ TVDS*PK 1+ TVDSPK 2+ YFLVGAGAIGCELLK 2+ LVLDSHIWAFK 3+

D

XXXXXXS*XXXX(K/R)

AQSGSDS(pS)PEPK 1+ EN(pS)PAAFPDR 1+ TVD(pS)PK 1+ DS(pS)VPETPDNER 1+ LFQLGPP(pS)PVK 1+ AY(pS)PEYR 1+ Ac-AEELVLER 1+ TLLEQLDDDQ 1+

Selective Isolation of Tryptic Phosphopeptides on 200-Å PolySULFOETHYL A

• from Gygi et al., PNAS 101 (2004) 12130-35 -

80 535 358 ERLIC SCX-IMAC

Nonredundant phosphopeptide id’s from gastric cancer cell line SNU5

• to identity as many as possible, use both methods -

Distribution of singly, doubly, triply and quadruply phosphorylated peptides by number of phosphate groups

1p (1578) 51% 2p (1115) 37% 3p (331) 11% ≥4p (21) 1% ≥4

A B

Phosphopeptide distribution from gastric cancer cell lines SNU5, SNU1, AGS, YCC1, & KatoIII (8% overlap)

Analysis of Phosphopeptides in a Tryptic Digest of Chicken Embryo Fibroblasts Infected with Marek’s Disease Virus

• Poster ThP-453, ASMS 2009: K.-Y. Chien, K. Blackburn, H.-C. S. Liu, & M.B. Goshe -

25 ERLIC fractions were collected & further processed via IMAC –

10-20x more phosphopeptides were identified via ERLIC + IMAC than with either method alone  Nonphosphopeptides suppress identification of phosphopeptides;

anything you do to get rid of them greatly increases phosphopeptide id’s * # phosphopeptides/ total peptides

pS 72% pT 22% pY 6%

Other methods: 1-2% pY sites. Why is % pY so high in ERLIC?

Phosphorylation sites, HeLa lysate digest

AIAAQLRY#T#R DDIYIRY#QS#FNNQSDLEK DKPRY#FIS#NNVSLMAAVTLIEES#C&SYK DSMVMHNSDPNLHLLAEGAPIDWGEEY#SNSGGGGSPS#PSTPESATLSEK EANTPY#PLYGPLNANLNAMDT#S#GQVNPEGR EGEEPTVY#SDEEEPK ELDY#AASLKY#EWNRR EQQT#QST#ELLNY#IIDTYNNIER ET#PY#AGAQVKQT#LSK FY#KGLLT#Y#FHYGMIYDLLSESEADAEK GGKAT#HAVRPTY#IVDPGET#PIYFLGR IVY#YY#T#DDRNK KVQERQYNPLPIEY#QLT#PY#EMLMDDIR RPAQSFWPDIC&KC&HY#T#HS#C&PLIK TQT#PPVS#PAPQPTEERLPSSPVY#EDAASFK TY#LEGEC&LELLR WFY#RHHY#DS#NHPRISSEVHIPLGDTR Y#GKELSM*VK Y#IIES#IENEDQLIEIYQTGS#DSMEIQNK Y#LM*TMIVGITSGFWIRS#GK Y#TEDIY#TLLEK

Sze et al. (2008): ERLIC is better than SCX-IMAC at identification of acidic phosphopeptides. Apparently that includes most pY sites. Here: 76% of phosphotyrosine sites were on peptides with > 1 phosphate EXAMPLE: Phosphotyrosine Peptides in HeLa Fraction 17

Y# = PhosphoTyr. T# = PhosphoThr. S# = PhosphoSer.

# id’s of phosphoproteins # Nonredundant phosphorylation sites Most of the pT’s and nearly all the pY’s are novel.

Either they’re unique to gastric cancer

• r they’re associated with peptides of

low abundance not detected by other methods. Question: Does the low phosphopeptide abundance reflect low protein abundance or low site occupancy?

Phosphopeptides in 5 gastric cancer cell lines via ERLIC & SCX-IMAC

(3021) (1211)

2144 (71%) 673 (22%) 204 (7%) 3021

20 40 60 time (min) 0.00 0.10 0.20 0.30 0.40 A210 p4 p1g+p1m p2 p3 p0 p5

H1.5 A

20 40 60 0.00 0.22 0.43 0.65 A 210 p1m p1g p2 p3 H1.5p0

An example:

CEX-HILIC of Histone H1.5 Phosphorylation Variants

COLUMN: PolyCAT A (204CT0510) GRADIENT: NaClO4 in 70% ACN (B. Sarg et al., JBC 281 (2006) 6573) INTERPHASE MITOSIS

p0 p1g ser17 ser172 p1m p2 ser(17,172) p3 ser(17,172,188) p4-1 ser(17,172,188) thr137 ser(17,172,188) p4-2 thr154 p5-1 ser(17,172,188) thr(10,137) ser(17,172,188) p5-2 thr(10,154)

INTERPHASE MITOSIS –

In this case, low abundance reflects low

• ccupancy of

the pT sites

Histone H1.5: Cell Cycle & Phosphorylation Sequence

Some Acidic Groups in Peptides

PHOSPHATE pKa ~ 2.1 SIALIC ACIDS pKa ~ 2.6 pKa ~ 2.7

ERLIC retains glycopeptides too

SAMPLE: Platelet membrane tryptic digest COLUMN: PolyWAX LP, 100x4.6-mm DETECTION: 295 nm GRADIENT: A) 20 mM Na-MePO3 + 70% ACN, pH 2.0 B) 200 mM TEAP + 60% ACN, pH 2.0 Nonglyco- peptides & asialoglyco- peptides (Sialylated?) Glycopeptides

(U. Lewandrowski et al., Clin. Proteom. 4 (2008) 25)

Simultaneous Isolation of Tryptic Phospho- and Glycopeptides via ERLIC

ex Mouse Brain Extract (1 mg) [Zhang et al., MCP 9 (2010) 635] Phospho- and glycopeptide content is ~ 30-90% in fractions 1-7. This compares favorably with “affinity” methods

CSF proteins Glycoproteins Glycopeptides Deglycosylated peptides Glycoproteins Peptides CSF Hydrazide resin Lectin affinity column Trypsin Trypsin PNGase F 2D LC-MS/MS 2D LC-MS/MS (or ----NH-N=CH- )

Affinity Chromatography of Glycoproteins & Glycopeptides

( courtesy Jing Zhang, Univ. of Washington )

# id’s in a replicate analysis

Identification of Unique Tryptic Glycopeptides in Mouse Brain Extract: ERLIC vs. Hydrazide Covalent Chromatography

• ERLIC is better -

[Zhang et al., MCP 9 (2010) 635]

Advantages of ERLIC for PTM isolation:

1) Affinity for -PO4, etc. is relatively weak and doesn’t dominate the chromatography. There is selectivity for the rest of the peptide. 2)  The enrichment and high resolution steps can be combined - much less work!

Distribution of peptides: Much more uniform in ERLIC than in SCX Basis of separation in ERLIC: pI of peptide (high to low) and polarity (low to high)

• both variables are complementary

to selectivity in RPC -

Rat Kidney Homogenate Tryptic Digest: Fractionation via ERLIC/RPC vs. SCX/RPC

ERLIC Gradient: A) 0.1% acetic acid in 90% ACN B) 0.1% formic acid in 30% ACN [NOTE: In A), -COOH are charged and selectivity for PTM’s is lost]

[P. Hao et. al., JPR 9 (2010) 3520-26]

PROTEOMICS:

Distribution of Components in Dimension #1 of 2-D HPLC

Uneven distribution; relatively few components identified in fractions 2 & 3 Ideal distribution; subsets (fractions) of equal size

ERLIC-RPC ERLIC-RPC SCX-RPC SCX-RPC

Protein id’s Unique Peptide id’s

The number of

identifications is significantly higher with ERLIC than with SCX. Also, ~ 2x more peptides id’d per protein with ERLIC (more rugged id’s)

Rat Kidney Homogenate Tryptic Digest: Fractionation via ERLIC/RPC vs. SCX/RPC

[P. Hao et. al., JPR 9 (2010) 3520-26]

Unique Unique Protein id’s Peptide id’s SCX: 3540 18,665 ERLIC: 4821 30,659 (32% more) (64% more)

ERLIC affords > 120% more id’s than does SCX with peptides that are: very basic

• r

very hydrophobic

Rat Kidney Homogenate Tryptic Digest: Fractionation via ERLIC/RPC vs. SCX/RPC

[P. Hao et. al., JPR 9 (2010) 3520-26]

5 10 15 20 25 30 35 40 45 50 mAU (214 nm) 500 1000 1500

Unmodified peptides Glycopeptides Phosphopeptides

SCX

Unmodified peptides Phosphopeptides

Min.

ERLIC

25 50 75 100 125

mAU (280 nm)

20 40 60 80 100

Fractionation of Rat Kidney Tryptic Peptides

ERLIC Gradient: A) 0.1% formic acid in 80% ACN B) 2% formic acid in 10% ACN [Compromise: Good selectivity for PTM’s but poor retention

• f unmodified peptides]

SCX: Fairly good selectivity for phosphopeptides; poor selectivity for (nonsialylated?) glycopeptides

ELECTROSTATIC MIXED-MODE EFFECTS IN HILIC

EXAMPLE: A tryptic peptide at pH  3.0

• +

+

• +

+

+ + +

+ A basic residue (N-terminus or C-terminal Arg- or Lys-)

A neutral, polar residue Cation-exchanger (SCX): Electrostatic attraction to basic residues; selectivity for other residues is tuned down. Neutral material: Pure hydrophilic interaction; selective for all polar residues. Anion-exchanger: Electrostatic repulsion of basic residues. They still promote retention through hydrophilic interaction, but selectivity for other residues is tuned up.

RESULT: ERLIC*

*ERLIC = Electrostatic Repulsion-Hydrophilic Interaction Chromatography

+ +

IS THIS MODEL CORRECT?

Minutes

5 10 15 20 25 30 35 40 45 50

mvolts

50 100 150 200

A, B & C A+P C+P D+P B+P 20  100 mM salt 70  10% ACN 260 nm 270 nm

F E

________________KEY_________________ A: SLYSSSPGGAYVTR (Vimentin(51-64)) B: SVNFSLTPNEIK (MAP 1B(1271-1282)) C: WWGSGPSGSGGSGGGK A+P: SLYSSS(p)PGGAYVTR B+P: SVNFSLT(p)PNEIK C+P: WWGSGPSGSGGS(p)GGGK D+P: GGAAGLGY(p)LGK E: WWGSGPS(p)GSGGS(p)GGGK F: WWGSGPSGS(p)GGS(p)GGGK

ERLIC of Phosphopeptides with Volatile Solvents

Column: PolyWAX LP (104WX0503) Flow rate: 1 ml/min Gradient: 0-5’: 0%B; 5-45’: 0-100% B; 45-50’: 100%B MP A: 20 mM NH4-Formate, pH 2.2, with 70% ACN MP B: [TOP] 100 mM NH4-Formate, pH 2.2, w. 64% ACN; [BOTTOM] 20 mM NH4-Formate, pH 2.2, w. 10% ACN Detection: A270 (blue) or A260 (red)

OBSERVATION: Retention time increases as the phosphate group gets closer to the C-terminus. Is this coincidence? Test it!

_ _ + + + + + HYPOTHETICAL ORIENTATION OF TRYPTIC PHOSPHOPEPTIDES

C O O

K

• PO4

+

• PO4

K

+

ERLIC & AEX SCX

_ _

O O C

PO4

• O4P

+

• 3.6
• 3.3
• 2.9
• 1.6
• 1.7

E

• 1.3
• 1.4
• 1.3
• 7.2
• 4.7
• 3.7
• 3.3
• 2.8

D

• 2.5
• 2.6
• 2.7

8.3 7.9 6.8 7.3 6.7

K

5.4 4.8 5.1 8.2 8.2 6.7 6.5

R

6.0 4.8 4.9 4.6

N-term N+1 N+2 N+3 N+4 C-3 C-2 C-1

Effect of position of charged residues from retention in SCX of 800,000 tryptic peptides, calculated via ANN [A.J. Alpert et al.,

• Anal. Chem. 82 (2010) 5253-59]

Peptides seem to be oriented in SCX with N-term. down; retention is especially sensitive to residues there. Basic residues: Affect retention anywhere in the peptide Acidic residues: Not so

SCX Fraction

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

#Unique Peptides

200 400 600 800

#Unique Peptides

100 200 300 400

Monophosphopeptides

 No extra basic residues  1 or more extra basic residues  N-Acetylated peptides  Phosphopeptides  Nonmodified peptides

Multiphosphor- ylated peptides Monophosphor- ylated peptides

Lys-N Phosphopeptides: Distribution in SCX Fractions

Sample: Lys-N digest of lysate of human embryonic kidney cells (HEK 293) Chromatography: PolySULFOETHYL A column; KCl gradient @ pH 2.7 Orientation is probably N-terminus down, so some phosphate groups are distant from the stationary phase and don’t induce repulsion better retention than N-Acet. peptides of the same net charge Peptides with extra basic residues have no well- defined orientation and can elute anywhere to the end of the gradient

No N- term. lysine

A.J. Alpert et al., Anal. Chem. 82 (2010) 5253-59]

Residue with Phosphate Group (N-Term. Lys = #0)

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 #Phosphopeptides in tallest bar SCX Fraction 19 SCX Fraction 20 SCX Fraction 21 SCX Fraction 22 SCX Fraction 23 SCX Fraction 24

Lys-N Phosphopeptides (no extra basic residues):

Distribution vs. Distance of Phosphate Group from the N-Terminal Lysine

These peptides are highly oriented. Retention increases with the distance of a phosphate group from the N-term.

A.J. Alpert et al., Anal. Chem. 82 (2010) 5253-59]

SCX Fraction

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

Monophosphopeptides  0 extra basic residues; K at C-term.  0 extra basic residues; R at C-term.  1 or more extra basic residues 2 PO4, 0 extra basic residues 2 PO4, 1 extra basic residue 3 PO4, 1 extra basic residue

# Phosphopeptides in tallest bar

Tryptic Phosphopeptides: Distribution in SCX

COLUMN: PolySULFOETHYL A, 200x2.1-mm; 5µm, 200Å SAMPLE: Tryptic digest of human embryonic kidney (HEK) 293 cells. 1 PO4, 0 basic res: Rigid orientation of binding; elution within a narrow range of the gradient 1 PO4 + extra basic res.: Other orientations of binding are possible and elution is possible throughout the gradient Additional PO4 + basic res.: The net charge determines the window of elution R vs. K at C-term.: No effect on retention

A.J. Alpert et al., Anal. Chem. 82 (2010) 5253-59]

VAVVRTPPKSPSSAK VAVVRpTPPKSPSSAK VAVVRpTPPKpSPSSAK VAVVRpTPPKpSPSpSAK

Regular HILIC can have trouble separating phosphorylation positional variants…

COLUMN: Luna HILIC SAMPLE: Tau(226-240) peptides GRADIENT: 5 mM NH4-formate, pH 3.2; 90% ACN for 10’, then 90-50% ACN over 15’, then hold @ 50% From: D. Singer et al., Anal. Chem. 82 (2010) 6409-14

…but ERLIC separates them readily

COLUMN: Luna-NH2 SAMPLE: Tau(226-240) peptides GRADIENT: 5 mM NH4-formate, pH 3.2; 90-50% ACN over 30’, then hold @ 50% VAVVRTPPKSPSSAK VAVVRpTPPKSPSSAK VAVVRTPPKpSPSSAK VAVVRTPPKSPSpSAK VAVVRpTPPKpSPSSAK VAVVRpTPPKSPSpSAK From: D. Singer et al., Anal. Chem. 82 (2010) 6409-14

CONCLUSIONS, ERLIC & Peptide Fractionation:

1) Selectivity for PTM’s: Need pH < 2.5 to uncharge COOH’s. Phosphopeptides & glycopeptides are both retained. 2) Good fractionation of all peptides: Start at pH > 3.5. Fractionation is by pI & polarity of the peptides 4) Peptides may be highly oriented during their migration. Orientation influences selectivity: The column can only interact with solute domains that can contact it.

ACKNOWLEDGEMENTS

Collaborators: Nanyang Technological University (Singapore): Siu Kwan Sze, Piliang Hao, Tiannan Guo, Xin Li, Jie Yang, James P. Tam, Huoming Zhang, Arnab Datta, Jung E. Park Institute of Medical Biology (Singapore): Sai K. Lim