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Strong cation-exchange high-performance liquid chromatography as a versatile tool for the characterization and purification of peptides
Ar%wTIcA CHIMICA ACTA
ELSEVIER
Analytica Chimica Acta 352 (1997) 21-30
Strong cation-exchange high-performance liquid chromatography as a versatile tool for the characterization and purification of peptides Dan L. Crimmins* Division of Laboratory Medicine, Department of Pathology, Washington University School of Medicine, Box 8118, 660 South Euclid Avenue, St. Louis, MO 63110, USA
Abstract Synthetic peptides play a major role in all areas of biomedical research. The ease with which virtually any sequence of both natural and unnatural amino acids can be synthesized is a primary reason for their popularity. Often, the final product not only contains the target peptide sequence but contaminating species that differ in subtle ways such as minor deletions, incomplete deprotected side chains, fragmentation at unexpected residues, and residue adducts as a consequence of improper resin cleavage. The investigator must then purify the desired target molecule from the crude mixture in order to conduct meaningful experiments. A widely practiced and highly successful method of analysis and purification utilizes reversed-phase highperformance liquid chromatography (RP-HPLC). An alternative chromatographic procedure uses strong-cation exchange (SCX)-HPLC. Here the primary mode of separation is the interaction of the peptides’ positive charges with the negatively charged functional moiety of the chromatographic support. At a mobile phase of pH 3, peptides are expected to possess a net positive charge as a result of protonation of carboxyl groups and C-terminus, leaving arginine, lysine, hi&dine and the Nterminus to contribute to the net positive charge. Thus, as a first approximation separation will be a monotonic function of differences in net positive charge. Considerable success has been realized with a sulfoethyl aspartamide SCX-HPLC support for the analysis and purification of well over 500 distinct synthetic and proteolytically derived peptide fragments. This short practical review will present examples of these separations with emphasis on synthetic peptides. For completeness, a section on SCX-HPLC of proteolytically derived peptides will also be presented. 0 1997 Elsevier Science B.V.
1. Introduction There is no single analytical technique that will consistently yield sufficient data to assess peptide sample purity, and for this reason, a combination of HPLC, N- and C-terminal sequence analysis, amino acid composition, capillary electrophoresis, and mass spectrometry is used. Recently however, improvements in instrument performance and data analysis *E-mail: [email protected]; fax: +l (314) 362 1461.
tel.: +l (314) 362 1622;
0003-2670/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SOOO3-2670(97)00091-3
for MS and MS-MS experiments have gone a long way toward realizing this goal. RP-HPLC is a popular chromatographic method for the analysis of peptides. The range of applications spans the characterization of synthetic peptides and peptide-mapping experiments. Alternate modes of chromatography play a useful role
in peptide characterization and in some instances provide data not observed in RP-HPLC. Ion-exchange (IEX)-HPLC is particularly well-suited for the analysis of peptides as most peptides possess net charges at a functional residue or an Nor C-terminus. Silica-based strong cation-exchange
22
D.L. Crimmins/Analytica
(SCX) solid phase material has certain advantages over the corresponding anion-based material and as such provides ideal resins for routine peptide characterization At a mobile phase pH near 3, peptide carboxylates are expected to be predominantly in a protonated form so that separation on SCX should result from the number of basic residues (Arg, His, and Lys) including the “free” N-terminus if present, in the peptide. The converse situation of deprotonating basic residues at high pH followed by silica-based anion-exchange HPLC is not possible due to the chemical instabiliy of silica at alkaline pH. Available polymeric-based anion exchangers would not remedy this situation for Arg-containing peptides because at the pH necessary to deprotonate this residue (->12) the functional group on the resin itself becomes deprotonated and therefore ineffective as an ion exchanger. In this review I summarize data on the use of SCXHPLC as a reliable and effective mode of chromatography for peptide analysis and characterization. Much of the data is taken from individual and collaborative efforts over the past ten years and includes synthetic peptide examples of SCX analysis for N-terminally blocked peptides, disulfidelinked peptides, and comparative evaluation of SCX-HPLC vs. capillary electrophoresis. In addition, SCX chromatograms of peptides derived from proteolysis ultimately used to identify post-translationally modified residues (e.g. phosphate) and assign cystine partners of disulfide-linked peptides are given.
2. Experimental
2.1. Reagents, chemicals, and solvents Peptides were provided by the Howard Hughes Medical Institute Core Protein/Peptide Facility at Washington University School of Medicine (St. Louis, MO) or purchased from Sigma (St. Louis, MO) or Peninsula Labs (Belmont, CA). Generally, the lyophilized material was reconstituted to a nominal concentration of 1 mg/ml with 0.1% (v/v) acetic acid and stored under a nitrogen blanket at 4°C. Where necessary, quantitation of peptide concentration was performed by amino acid analysis using either
Chimica Acta 352 (1997) 21-30
phenylisothiocyanate [ 1,2] or 6-aminoquinolyl-Nhydroxysuccinimidyl carbamate [3] precolumn derivatization of the hydrolyzate with reagents, standards, and columns obtained from Pierce Chemical (Rockford, IL) and Waters (Milford, MA). Disulfide-linked synthetic peptides were prepared via a thiol-disulfide interchange reaction [4,5] or derived from proteolysis of unreduced proteins [6,7]. HPLC grade solvents acetonitrile (MeCN) and methanol were Burdick and Jackson products from Baxter (McGaw Park, IL), and 85% (w/v) phosphoric acid (HsPOd) from Fisher (St. Louis, MO). A Mini-Q (Millipore, Boston, MA) apparatus was used to generate high-quality water by sequential passage through ion-exchange and carbon canisters with 0.2 pm final filter. The enzymatic procedures to generate proteolytically derived peptide fragments can be found in the original references [2,6-91. Additional materials were obtained from local suppliers as the highest quality available. 2.2. HPLC Waters equipment was used throughout comprising two 5 10 pumps, a 680 gradient controller, a 710 WISP autosampler, a 490 four-channel detector, and a 1122 column temperature controller. Data acquisition was accomplished with Nelson Analytical (now PE-Nelson, Cupertino, CA) 700 series A/D boxes and 4400 series software [ 1,2]. The sulfoethyl aspartamide column (manufactured under the trade name PolySULFOETHYL Aspartamide by PolyLC, Columbia, MD) was 300-A, 5-p, 200 mmx4.6 mm material and purchased from the NEST group (Southborough, MA). The column was developed with a linear AB type gradient at 1 ml/min and 28°C as either 0 to 60% B in 60 minutes or 0 to 100% B in 60 minutes with mobile phase A = 5 mM sodium phosphate pH 3,25% (v/v) MeCN and mobile phase B = 5 mM phosphate, 500 mM NaCl pH 3, 25% (v/v) MeCN. Where desired, fractions were usually collected at 0.5 to 1.0 minute intervals over the entire gradient. Selected fractions were analyzed by amino acid analysis, N-terminal amino acid sequencing, and rechromatography on RP-HPLC for desalting [ 1,2]. In the later stages of this work, mass spectrometry was used to assess fraction purity [6,7].
D.L. Crimmins/AnalyricaChimicaActa 352 (1997) 21-30
+6
+5
+2
t
u
i I
I 10.00
I 20.00
I 30.00
I 40.00
+7
I!
I 50.00
I 60.00
Min.
Fig. 1. SCX chromatogram of a mixture of seven peptides ranging in net nominal charge at pH 3 of +l to +7. In order of increasing retention time, the peptide mixture comprised YPFVEPI, VLPRPTITM, KEKLIAPVA, DYGGIKKIRLPSDDVC, RQAGDDFSRRYRGDFAE, YGGFLRRIRPKLK, and KPVGKKRRPVKYP, 205 mVolts-full scale. From [I] with permission.
3. Results and discussion 3.1. Monotonic
elution ofpositively
chargedpeptides
Fig. 1 displays the chromatographic elution profile of a mixture of synthetic peptides ranging in net nominal positive charge at pH 3 of $1 to $7 using a 0 to 100% B gradient [l]. These elution characteristics on the sulfoethyl aspartamide column were independently reported in a peer-reviewed journal by Crimmins et al. [l] and Alpert and Andrews [lo]. Both groups realized the versatile nature of this column for the characterization and purification of peptides. In one of the original reports [l] over 50 unmodified peptides ranging in net nominal charge at pH 3 from +l to +7 and 15 modified peptides of charges +l to +3 were shown to obey monotonic elution profiles. Several important observations from these studies were stated including the potential for a mixed-mode separation mechanism, facile resolution of N-terminally blocked peptides from unmodified counterparts, and successful resolution on SCXHPLC of charge variants not achievable by standard RP-HPLC. The overall results were analyzed in more detail in an effort to quantitatively describe elution characteristics on this column [ 1 l] as well as comparing the SCX separation with another cation-exchange
23
matrix [ 121. General applications of sulfoethyl aspartamide SCX-HPLC peptide purification have been presented [13]. 3.2. Facile analysis of N-terminally via SCX-HPLC
blocked peptides
Figs. 2-4 present examples of the chromatographic resolution of blocked N-terminal peptides from ‘free’ N-terminal peptides as a direct comparison of SCXHPLC vs. RP-HPLC. Fig. 2 panels B and D illustrate the excellent resolution of N-terminally blocked peptides labeled II and IV, in this case acetylated, from their respective ‘free’ N-terminal peptides I (RRPYIL) and III (DRVYIHPFHLLVYS) [ 11. A positive charge is consumed by acetylation. Thus the blocked species have a net positive charge of one less than the unblocked parent molecules and so elute earlier on SCX-HPLC. For comparison, panels A and C show the corresponding RP-HPLC pattern for each blockedunblocked peptide pair. Here the elution order is reversed as the acetylated species are less polar (more hydrophobic) than the unblocked peptides. Another example of this phenomenon is shown in Fig. 3 [ 141. Pyroglutamic acid is a cyclized glutamine
II I
10.00
I 20.00
II I 30.00
. I 40.00
I 50.00
Min.
Fig. 2. RP and SCX chromatograms of N-terminal acetylated peptides. (A) RP chromatogram of peptide I = RRPYIL and peptide II = N-acetyl-RRPYIL, 140 mVolts-full scale. (B) SCX chromatogram of peptide pair I/II at 165 mVolts-full scale. (C) RP chromatogram of peptide III = DRVYIHPFHLLVYS and peptide IV = N-acetyl-DRVYIHPFHLLVYS, 555 mVolts-full scale. (D) SCX chromatogram of peptide pair IWIV at 235 mVolts-full scale. From [I] with permission.
D.L. Crimmins/Analytica Chimica Acta 352 (1997) 21-30
24
1l
pGlu
7
5
I
Q i; i
a
I
A
A
I
II
I
I RP
pGlu
l
I_-
I
,
I ____-
RP
A
L
I
I
IO
20
I
L
-
30 TIME (mid
I
40
I
50
Fig. 3. SCX and RP chromatograms of lutenizing hormone releasing factor, pEHWSYGLRPG(NHs), containing an N-terminal pyroglutamic acid (pE) and C-terminal amide (NH*). (A) SCX chromatogram of untreated peptide, 750 mVolts-full scale. (B) SCX chromatogram of PGAPase treated peptide, 375 mVolts-full scale. (C) RP chromatogram of untreated peptide, 725 mVolts-full scale. (D) RP chromatogram of PGAP’ase treated peptide, 725 mVolts-full scale. From [14] with permission.
I IO
I I 20
,L%
I
30 TIME(min)
RP I 40
I 50
j
Fig. 4. SCX and RP chromatograms of C-terminal Human Interleukin-2 Receptor peptide, QRRQRKSRRTI. (A) SCX chromatogram of stock peptide in water showing two N-terminal species, the native molecule (Gln) and the blocked derivative (pGlu), 70 mVolts-full scale. The arrow indicates the elution position of a new peak after PGAP’ase treatment of the stock peptide solution. (B) RP chromatogram of the stock peptide, 185 mVolts-full scale. From [14] with permission.
D.L. Crinunins/Analytica
residue minus one molecule of water and when present as the N-terminus of a peptide is refractory to Edman sequencing chemistry. The blocked residue can be removed with the aid of an enzyme, Pyroglutamate Aminopeptidase (PGAP’ase), yielding an N- 1 peptide with a free N-terminus. One expects then, that the deblocked peptide will elute later on SCX-HPLC than the pyroglutamyl-peptide since deblocking effectively generates an additional positive charge on the molecule. This is precisely what is observed in panels A and B of Fig. 3 [14]. Again the elution order is reversed on RP-HPLC as demonstrated in panels C and D but with less resolution under standard gradient conditions than for standard SCX-HPLC analysis. There are instances where RP-HPLC does not resolve a blocked-unblocked peptide pair but SCXHPLC does. Such an example is displayed in Fig. 4 as panels A and B [ 141. The stock C-terminal portion of Human Interleukin-2 Receptor peptide with sequence QRRQRKSRRTI apparently contains both the target peptide and a cyclized derivative as pyroglutamic acid. This was surmised from the isolation and subsequent analysis of the two SCX-HPLC peaks shown in panel A. Only the later eluting peak labeled ‘Gln’ gave Nterminal sequence following Edman degradation and treatment of the mixture with PGAP’ase resulted in loss of the peak labeled ‘pGlu’ (pyroglutamic acid) but not peak ‘Gln’. Importantly, the appearance of a new peak with greater retention time was clearly evident (indicated by an arrow) being consistent with removal of the cyclized N-terminal pyroglutamic acid residue and increase in net positive charge. Interestingly, the stock peptide solution showed no such heterogeneity when analyzed via RP-HPLC, panel B, convincingly demonstrating that SCX-HPLC can provide chromatographic separation not achievable by RP-HPLC. 3.3. Disulfide-linked following
dimers are easily isolated
SCX-HPLC
Homo- or hetero-peptide dimers covalently joined by one or more disulfide bonds are produced in a variety of ways. For instance, after cleavage of the cysteine-containing peptide from the solid-phase resin, from enzymatic/chemical cleavage of unreduced proteins, as an artifact generated during purification of cysteine-containing peptide fragments from various sources, and through synthesis to study
Chimica Acta 352 (1997) 21-30
25
for example avidity, receptor binding, enzymatic inhibition, and structural and conformational properties of dimeric vs. monomeric peptides. However produced, the homo- or hetero-dimeric species should be more positively charged than the corresponding individual monomeric constituents. For homo-dimers the charge is simply twice that of the monomeric chain and for hetero-dimers the charge is the sum of the individual peptide chains. Analytically, several methods are available to detect disulfide-linked peptides ([4], and references therein) including two-dimensional electrophoresis, pre-column sample modification with sulfhydryl reagents, post-column analysis via mass spectrometry or electrochemical detection, N-terminal Edman sequencing, and recently, in situ linkage assignment via Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry [7,15]. Generally, a combination of techniques is necessary to absolutely determine disulfide bond partners. Preparatively, three distinct chromatographic modes seem well-suited to isolate the dimer from the monomer. Size-exclusion (SE)-HPLC should separate the desired components but with attendant sample dilution and the caveat that the two components might not be resolved if the resulting threedimensional shape of the dimer is not that different from the monomer [ 161. This follows because the primary separation mechanism of SE-HPLC is governed by the shape/conformation of the analytes and strictly speaking, not the molecular weight. RP-HPLC provides a predictable elution order for the dimermonomer peptide pair because the dimer is expected to be more hydrophobic and thus retained longer on the column. Interestingly, association of two monomeric species to form a dimer can frequently mask hydrophobic residues from interaction with the column. Mechanistically, the peptide-peptide hydrophobic interaction is apparently greater than the peptidecolumn hydrophobic forces, so in this case the disulfide-linked dimer elutes earlier than the monomeric components [17]. As stated above, the net positive charge of the disulfide-linked dimer will be greater than either of the individual constituents of the monomer [4,5,7] which immediately suggests SCX-HPLC as a preparative mode of purification. As an example, two synthetic peptides, a 1Cmer ALLETYCATPAKSE with an internal position
26
D.L. Crimmins/Analytica
Chimica Actn 352 (1997) 21-30
sex
A
::I:‘1 3
Gfl
2
I
as
d
ST
10
S Y
/
ClBRP
.I
E
0*
-‘
i i
PA
c.
i-
Ak T
:
A/j
d 30 min
50
Fig. 5. HPLC purification of a Disulfide-linked Heterodipeptide. (A) SCX-HPLC of the thiol-disulfide interchange mixture of ALLETYCATPAKSE, A,-C7-Et_,, and GRGDSPC, Gt-C,. Peaks 1 through 4 were assigned from appropriate homodipeptide reactions: 1 = 2-nitro-5-thiobenzoic acid:G,-C,, 2 = 2-nitro-5-benzoic acid:A,-C,-Et4, 3 = Gt-C7:GI-C,, and 4 = At-C,-Et4:AI-C7-E14. The peak marked with a star was the desired heterodipeptide, A,CT-E,d:Gt-C,, 1750 mVolts-full scale. (B) RP-HPLC desalting of SCX purified heterodipeptide, 1750 mVolts-full scale.
cysteine and a 7-mer GRGDSPC with a C-terminal cysteine, were disulfide linked following a thiol-disulfide interchange reaction with 5,5’-dithiobis(2_nitrobenzoic acid) [4,5,7]. The chromatographic analysis is profiled in Fig. 5 with panel A representing SCXHPLC of the reaction mixture and panel B, desalting via RP-HPLC. Peaks labeled 1 and 2 in panel A are mixed-disulfide adducts with individual peptide monomers while peaks 3 and 4 are homo-dimers ascertained from independent reactions and chromatography (data not shown). The starred peak was desalted by RP-HPLC (panel B) and subjected to a battery of analytical procedures for confirmation of structure. Amino acid compositional analysis (expected/observed values) as residues per mole of dipeptide was: D (l/1.0), E (2/2.1), S (2/2.0), G (2/2.2), R (l/1.0), T (2/2.0), A (3/3.0), P (2/1.9), Y (l/0.95), C (2/net determined), L (2/l.@, and K (110.95). The correlation was good and indicates that the molar composition of the sample was consistent with the disulfide-linked peptide. Fig. 6 is a composite for the N-terminal sequence analysis of the dipeptide. As expected, the first six cycles show two residues were released corresponding to the sequence of the individual peptides. For example, at cycle 1 the two amino acids identified were A and G corresponding to the N-
Fig. 6. N-terminal Sequence Analysis of Disultide-linked Heterodipeptide, At-C7-Et4:G1-C7. S, standard mixture of phenyhhiohydantoin amino acids indicated by the usual one-letter code with X= reagent by-products. The time axis is shifted slightly to the left to avoid display overlap from cycle 1. Note, two residues were assigned in sequencing cycles 1 through 6, one residue was assigned as cystine (C,) in cycle 7, only one residue was assigned in sequencing cycles 8 through 14, and no residue was assigned in cycle 15 as expected, 40 mVolts-full scale each. Approximately 300 pmoles of sample was loaded.
terminal residue of each constituent peptide monomer. At cycle 7, a peak presumed to be diphenylthiohydantoin cystine is observed (C,) [ 181 after which cycles 8 through 14 show only residues attributable to the 14-mer peptide. Mass spectrometry gave M+ equal to 2188 vs. an expected value of 2187 along with the masses of the individual monomeric molecules [7]. There is little doubt then that the starred peak in panel A of Fig. 5 is the target hetero-dimer. Ten distinct synthetic homo-/hetero-dimers have been successfully isolated using SCX chromatography [4,5,7]. 3.4. Comparison
of SCX-HPLC
capillary electrophoresis
and free-solution
(FSCE) for peptide
analysis
Two reports have been published on the comparative analysis of SCX-HPLC vs. FSCE [19,20]. Data from each shows the elution order for mixtures of peptides is reversed on FSCE at pH 2 or 3 from that of SCX at pH 3. This is in complete accord with one’s intuition as the more positively charged molecules will migrate faster on FSCE and therefore elute earlier than
D.L. Crimmins/Analytica
Chimica Acta 352 (1997) 21-30
21
Fig. 7. SCX-HPLC and FSCE of synthetic peptides with C-terminal and methionine modifications, and truncated species. SCX-HPLC at pH 3, unprimed panels A, 1250 mVolts-full scale, B and C, 500 mVolts-full scale. FSCE at pH 3, single-primed panels, all 325 mVolts-full scale. FCSE at pH 2, double-primed panels, all 325 mVo1t.vfull scale. From [19] with permission.
less positive peptides. The electrophoretic and chromatographic techniques are thus complementary. Fig. 7 compares the SCX-HPLC profile of synthetic mixtures at pH 3 (unprimed panels) with FSCE at pH 3 (single primed panels) and pH 2 (double primed panels) [ 191. The peptide mixtures reveal another feature of the sulfoethyl aspartamide column not previously discussed, that in addition to charge-influenced separation, polarity can contribute to resolution [ 1,21-231. Panels A through C illustrate the dramatic effect of polarity, here at the C-terminus, i.e., ‘free acid’ (OH) to amide [(Nl&)], on retention properties. In all cases the more polar acid variant is retained less than the corresponding amide. Note that resolution is
better for FSCE at pH 3 (A’ through C’) compared to pH 2 (A’ through C’) but requires a longer run time. Although it is now feasible to ‘preparatively’ isolate analytes from FSCE by multiple runs using computer controlled fraction overlay it is still rather tedious and little material is actually generated. For these reasons, SCX-HPLC is preferred for the isolation of sufficient quantities of synthetic peptides. 3.5. Peptides derived by proteolysis The versatile nature of the sulfoethyl aspartamide column is further exemplified by the excellent resolution of peptide fragments generated from peptide
28
D.L. Crimmins/Analytica Chimica Acta 352 (1997) 21-30
Fig. 8. SCX and RP chromatograms following V8 protease digestion of @-Casein. (A) SCX-HPLC of digest, 140 mVolts-full scale. The numbered peaks were subjected to ammo acid analysis and N-terminal sequencing to identify the peptide fragments. The elution position of the identified peptide fragments vs. calculated net charge at pH 3 was monotonic (data not shown). (B) RP-HPLC of same digest, 250 mVolts-full scale. From [2] with permission.
mapping experiments. Proteolysis with for instance Staphylococcus aureus V8 protease, which cleaves on the C-terminal side of glutamic and aspartic acid residues, provides a predictable elution pattern for known substrates. Separation then, to a first approximation, should be a function of the number of basic residues present in the generated fragments [2,24]. Fig. 8 compares the SCX (panel A) and RP (panel B) chromatograms for a V8 digest of P-Casein [2]. The numbered peaks in the SCX profile were sequenced [2] and the experimental elution of these identified peptide fragments were consistent with the monotonic nature of charge vs. retention time reported in Fig. 1. Representative peptide mapping applications for this column include the selective isolation of C-terminal and blocked amino terminal viral proteins following proteolysis [25], isolation of two-dimensional Asp-N proteolytic peptides [26], and purification of UDPglucose dehydrogenase peptides for N-terminal sequence analysis [27]. Phosphorylation site determination for Troponin T [28], Insulin Receptor Substrate IRS-l [8], and Calmodulin [9] utilized SCX-HPLC for isolation of the labeled peptide prior to additional analysis. The retention time of the 32P-labeled peptide on this column was used to eliminate one candidate proteolytic fragment from another, thereby simplifying the
Fig. 9. HPLC purification of selected disultide-linked human von Willebrand Factor proteolytic peptides. (A) RP-HPLC of Sraphylococcus aumus VUtrypsin proteolyzed human von Willebrand Factor. Fractions were collected at 0.5 min intervals with fraction 111 (arrow) and fractions 132-137 (pooled for Asp-N digestion) taken for further analysis, 1350 mVolts-full scale. (B) SCX-HPLC of fraction 111 from A. Fractions were collected at 1.0 min intervals with fraction 37 (arrow) taken for further analysis, 90 mVolts-full scale. (C) RP-HPLC of the Asp-N digest of pooled fractions 132-137 from A. Fractions were collected at 0.5 minute intervals with fraction 110 (arrow) taken for further analysis, 1350 mVolts-full scale. (D) SCX-HPLC of fraction 110 from C. Fractions were collected at 1.0 min intervals with fraction 23 (arrow) taken for further analysis, 230 mVolts-full scale. From [7] with permission.
radiosequencing results and in combination, provided positive assignment of the phosphorylation site [8,9]. Dimeric synthetic peptides are easily identified on SCX-HPLC as shown above so it was logical to attempt to isolate disulfide-linked peptide fragments from enzymatic/chemical digests. These results were described in general for tryptic digests [29] and in particular for human complement component C3b [30] and human von Willebrand Factor [6]. The latter publication clearly showed the versatile nature of the
D.L. Crimmins/Adytica
Chimica Acta 352 (1997) 21-30
sulfoethyl aspartamide column as an indispensable aid in protein/peptide chemistry applications. Fig. 9 displays the two-dimensional chromatographic isolation of a disulfide-linked tetrapeptide (panels A and B) and a disulfide-linked tripeptide (panels C and D) [6]. It was relatively straightforward in this case to choose the appropriate fractions for further analysis because the later eluting fragments were either very basic monomeric peptides or covalently joined multimeric peptides. Edman sequencing and mass spectrometry were used to determine which possibility was the correct one. In fact, since the goal here was to assign disulfide partners in the ‘T2’ domain of human von Willebrand Factor, SCX-HPLC was used as a positive selection screening technique to generate a manageable number of candidates for subsequent analysis [6]. An interesting variation was presented for E-(y-Glutamyl)lysyl cross-Iinks in Transglutaminase-modified Human Plasminogen [31]. Again, the covalently joined dimeric peptide elutes later on SCX-HPLC due to an increase in net positive charge at pH 3. In these experiments [31] this covalent dimer actually appeared as a new SCX peak for the Transglutaminase treated protein compared to the untreated control and was thus easily isolated and structurally identified.
4. Conchsions The author has successfully used sulfoethyl aspartamide SCX columns as a versatile research tool from 1987 to the present. Many different types of samples have been analyzed during this time yet all separations in one way or another exploit differences in nominal net positive charge at pH 3 as the primary mechanism of chromatographic retention. Thus, when either consumption or generation of positive charge occurs during a chemical reaction, SCX-HPLC ought to be given prime consideration as a valuable characterization and purification technique. The interested reader is encouraged to seek other reviews on ion-exchange HPLC of peptides [11,32,33].
References [l] D.L. Crimmins, Chromatogr.
.I. Go&a, R.S. Thoma, 443 (1988) 63.
B.D. Schwartz,
J.
29
VI D.L. Crimmins, R.S. Thoma, D.W. McCourt, B.D. Schwartz, Anal. Biochem. 176 (1989) 255. 244 (1997) [31 D.L. Crimmins, R. Cherian, Anal. B&hem. 407. [41 D.L. Crimmins, Pept. Res. 2 (1989) 395. VI D.L. Crinunins, in B.M. Dunn, M.W. Pennington (eds.), Peptide Analysis Protocols (Methods in Molecular Biology, Humana Press, Totowa, NJ, Vol 36, 1994, p. 53. VI 2. Dong, R.S. Thoma, D.L. Crimmins, D.W. McCourt, E.A. Tuley, J.E. Sadler, J. Biol. Chem. 269 (1994) 6753. [71 D.L. Crimmins, M. Saylor, J. Rush, R.S. Thoma, Anal. Biochem. 226 (1995) 355. M.G. Meyers, Jr., R.S. Thoma, D.L. PI M.J. Tanasijevic, Crimmins, M.F. White, D.B. Sacks, J. Biol. Chem. 268 (1993) 18157. R.S. Thoma, D.B. Sacks, [91 J.L. Joyal, D.L. Crimmins, Biochem. 35 (1996) 6267. J. Chromatogr. 443 (1988) t101 A.J. Alpert, P.C. Andrews, 85. [Ill A.J. Alpert, in CT. Mant, R.S. Hodges (eds.), HighPerformance Liquid Chromatography of Peptides and Proteins: Separation, Analysis and Conformation, CRC Press, Boca Raton, FL, 1991, p. 187. [I21 T.W. Loren Burke, C.T. Mant, J.A. Black, R.S. Hodges, J. Chromatogr. 476 (1989) 377. [I31 P.C. Andrews, Pept. Res. 1 (1988) 93. 1141 D.L. Crimmins, D.W. McCourt, B.D. Schwartz, Biochem. Biophys. Res. Commun. 156 (1988) 910. [W S.D. Patterson, V. Katta, Anal. Chem. 66 (1994) 3727. [1’51 D.L. Crimmins, M.E. Holtzer, unpublished observations. [I71 R.S. Hodges, P.D. Semchuk, A.K. Taneja, CM. Kay, J.M.R. Parker, C.T. Mant, Pept. Res. 1 (1988) 19. [I81 P. HGjrup, S. Magnusson, Biochem. J. 245 (1987) 887. u91 Guarino, Phillips, Amer. Lab., 23 (1991) 68. [201 D.L. Crimmins in R. Angeletti (ed.), Techniques in Protein Chemistry III, Academic Press, San Diego, CA, 1992, p. 171. [211 A.J. Alpert, J. Chromatogr. 499 (1990) 177. 548 [221 B.-Y. Zhu, CT. Ma.nt, R.S. Hodges, J. Chromatogr. (1991) 13. 594 ~231 B.-Y. Zhu, CT. Mant, R.S. Hodges, J. Chromatogr. (1992) 75. ~241 P. Iadarola, M.C. Zapponi, L. Minchiotti, M.L. Meloni, M. Galliano, G. Ferri, J. Chromatogr. 512 (1990) 165. t251 J.J. Gorman, B.J. Shiell, J. Chromatogr. 646 (1993) 193. [261 T.D. Schlabach, J.C. Colbum, R.J. Mattaliano, S. Yuen, in T. Hugli (ed.), Techniques in Protein Chemistry, Academic Press, San Diego, CA, 1989, p. 497. ~271 J. Hempel, J. Perozich, H. Romovacek, A. Hinich, I. Kuo, D.S. Feingold, Prot. Sci. 3 (1994) 1074. [281 K. Swiderek, K. Jaquet, H.E. Meyer, C. Schachtele, F. Hofmann, L.M.G. Heilmeyer, Jr., Eur. J. Biochem. 190 (1990) 575. WI P.C. Andrews, in J. Villafranca (ed.), Current Research in Protein Chemistry: Techniques, Structure, and Function, Academic Press, San Diego, CA, 1990, p. 95. [301 K. Dolmer. L. Sottnm-Jensen. FEBS Lett. 315 (1993) 85.
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[31] E. Bendixen, PC. Harpel, L. Sottrup-Jensen, J. Biol. Chem. 270 (1995) 17929. [32] A. Fallon, R.F.G. Booth, L.D. Bell, in R.H. Burdon, PH. vanKnippenberg (eds.), Applications of HPLC in Biochemistry, Laboratory Techniques in Biochemistry and Molecular
Chimica Acta 352 (1997) 21-30 Biology, Elsevier, Amsterdam, Netherlands, Vol 17, 1987, p. 43. [33] M.P. Henry, in R.W.A. Oliver (ed.), HPLC of Macromolecules, A Practical Approach, IRL Press, New York, 1989, p. 191.
ELSEVIER
Analytica Chimica Acta 352 (1997) 21-30
Strong cation-exchange high-performance liquid chromatography as a versatile tool for the characterization and purification of peptides Dan L. Crimmins* Division of Laboratory Medicine, Department of Pathology, Washington University School of Medicine, Box 8118, 660 South Euclid Avenue, St. Louis, MO 63110, USA
Abstract Synthetic peptides play a major role in all areas of biomedical research. The ease with which virtually any sequence of both natural and unnatural amino acids can be synthesized is a primary reason for their popularity. Often, the final product not only contains the target peptide sequence but contaminating species that differ in subtle ways such as minor deletions, incomplete deprotected side chains, fragmentation at unexpected residues, and residue adducts as a consequence of improper resin cleavage. The investigator must then purify the desired target molecule from the crude mixture in order to conduct meaningful experiments. A widely practiced and highly successful method of analysis and purification utilizes reversed-phase highperformance liquid chromatography (RP-HPLC). An alternative chromatographic procedure uses strong-cation exchange (SCX)-HPLC. Here the primary mode of separation is the interaction of the peptides’ positive charges with the negatively charged functional moiety of the chromatographic support. At a mobile phase of pH 3, peptides are expected to possess a net positive charge as a result of protonation of carboxyl groups and C-terminus, leaving arginine, lysine, hi&dine and the Nterminus to contribute to the net positive charge. Thus, as a first approximation separation will be a monotonic function of differences in net positive charge. Considerable success has been realized with a sulfoethyl aspartamide SCX-HPLC support for the analysis and purification of well over 500 distinct synthetic and proteolytically derived peptide fragments. This short practical review will present examples of these separations with emphasis on synthetic peptides. For completeness, a section on SCX-HPLC of proteolytically derived peptides will also be presented. 0 1997 Elsevier Science B.V.
1. Introduction There is no single analytical technique that will consistently yield sufficient data to assess peptide sample purity, and for this reason, a combination of HPLC, N- and C-terminal sequence analysis, amino acid composition, capillary electrophoresis, and mass spectrometry is used. Recently however, improvements in instrument performance and data analysis *E-mail: [email protected]; fax: +l (314) 362 1461.
tel.: +l (314) 362 1622;
0003-2670/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SOOO3-2670(97)00091-3
for MS and MS-MS experiments have gone a long way toward realizing this goal. RP-HPLC is a popular chromatographic method for the analysis of peptides. The range of applications spans the characterization of synthetic peptides and peptide-mapping experiments. Alternate modes of chromatography play a useful role
in peptide characterization and in some instances provide data not observed in RP-HPLC. Ion-exchange (IEX)-HPLC is particularly well-suited for the analysis of peptides as most peptides possess net charges at a functional residue or an Nor C-terminus. Silica-based strong cation-exchange
22
D.L. Crimmins/Analytica
(SCX) solid phase material has certain advantages over the corresponding anion-based material and as such provides ideal resins for routine peptide characterization At a mobile phase pH near 3, peptide carboxylates are expected to be predominantly in a protonated form so that separation on SCX should result from the number of basic residues (Arg, His, and Lys) including the “free” N-terminus if present, in the peptide. The converse situation of deprotonating basic residues at high pH followed by silica-based anion-exchange HPLC is not possible due to the chemical instabiliy of silica at alkaline pH. Available polymeric-based anion exchangers would not remedy this situation for Arg-containing peptides because at the pH necessary to deprotonate this residue (->12) the functional group on the resin itself becomes deprotonated and therefore ineffective as an ion exchanger. In this review I summarize data on the use of SCXHPLC as a reliable and effective mode of chromatography for peptide analysis and characterization. Much of the data is taken from individual and collaborative efforts over the past ten years and includes synthetic peptide examples of SCX analysis for N-terminally blocked peptides, disulfidelinked peptides, and comparative evaluation of SCX-HPLC vs. capillary electrophoresis. In addition, SCX chromatograms of peptides derived from proteolysis ultimately used to identify post-translationally modified residues (e.g. phosphate) and assign cystine partners of disulfide-linked peptides are given.
2. Experimental
2.1. Reagents, chemicals, and solvents Peptides were provided by the Howard Hughes Medical Institute Core Protein/Peptide Facility at Washington University School of Medicine (St. Louis, MO) or purchased from Sigma (St. Louis, MO) or Peninsula Labs (Belmont, CA). Generally, the lyophilized material was reconstituted to a nominal concentration of 1 mg/ml with 0.1% (v/v) acetic acid and stored under a nitrogen blanket at 4°C. Where necessary, quantitation of peptide concentration was performed by amino acid analysis using either
Chimica Acta 352 (1997) 21-30
phenylisothiocyanate [ 1,2] or 6-aminoquinolyl-Nhydroxysuccinimidyl carbamate [3] precolumn derivatization of the hydrolyzate with reagents, standards, and columns obtained from Pierce Chemical (Rockford, IL) and Waters (Milford, MA). Disulfide-linked synthetic peptides were prepared via a thiol-disulfide interchange reaction [4,5] or derived from proteolysis of unreduced proteins [6,7]. HPLC grade solvents acetonitrile (MeCN) and methanol were Burdick and Jackson products from Baxter (McGaw Park, IL), and 85% (w/v) phosphoric acid (HsPOd) from Fisher (St. Louis, MO). A Mini-Q (Millipore, Boston, MA) apparatus was used to generate high-quality water by sequential passage through ion-exchange and carbon canisters with 0.2 pm final filter. The enzymatic procedures to generate proteolytically derived peptide fragments can be found in the original references [2,6-91. Additional materials were obtained from local suppliers as the highest quality available. 2.2. HPLC Waters equipment was used throughout comprising two 5 10 pumps, a 680 gradient controller, a 710 WISP autosampler, a 490 four-channel detector, and a 1122 column temperature controller. Data acquisition was accomplished with Nelson Analytical (now PE-Nelson, Cupertino, CA) 700 series A/D boxes and 4400 series software [ 1,2]. The sulfoethyl aspartamide column (manufactured under the trade name PolySULFOETHYL Aspartamide by PolyLC, Columbia, MD) was 300-A, 5-p, 200 mmx4.6 mm material and purchased from the NEST group (Southborough, MA). The column was developed with a linear AB type gradient at 1 ml/min and 28°C as either 0 to 60% B in 60 minutes or 0 to 100% B in 60 minutes with mobile phase A = 5 mM sodium phosphate pH 3,25% (v/v) MeCN and mobile phase B = 5 mM phosphate, 500 mM NaCl pH 3, 25% (v/v) MeCN. Where desired, fractions were usually collected at 0.5 to 1.0 minute intervals over the entire gradient. Selected fractions were analyzed by amino acid analysis, N-terminal amino acid sequencing, and rechromatography on RP-HPLC for desalting [ 1,2]. In the later stages of this work, mass spectrometry was used to assess fraction purity [6,7].
D.L. Crimmins/AnalyricaChimicaActa 352 (1997) 21-30
+6
+5
+2
t
u
i I
I 10.00
I 20.00
I 30.00
I 40.00
+7
I!
I 50.00
I 60.00
Min.
Fig. 1. SCX chromatogram of a mixture of seven peptides ranging in net nominal charge at pH 3 of +l to +7. In order of increasing retention time, the peptide mixture comprised YPFVEPI, VLPRPTITM, KEKLIAPVA, DYGGIKKIRLPSDDVC, RQAGDDFSRRYRGDFAE, YGGFLRRIRPKLK, and KPVGKKRRPVKYP, 205 mVolts-full scale. From [I] with permission.
3. Results and discussion 3.1. Monotonic
elution ofpositively
chargedpeptides
Fig. 1 displays the chromatographic elution profile of a mixture of synthetic peptides ranging in net nominal positive charge at pH 3 of $1 to $7 using a 0 to 100% B gradient [l]. These elution characteristics on the sulfoethyl aspartamide column were independently reported in a peer-reviewed journal by Crimmins et al. [l] and Alpert and Andrews [lo]. Both groups realized the versatile nature of this column for the characterization and purification of peptides. In one of the original reports [l] over 50 unmodified peptides ranging in net nominal charge at pH 3 from +l to +7 and 15 modified peptides of charges +l to +3 were shown to obey monotonic elution profiles. Several important observations from these studies were stated including the potential for a mixed-mode separation mechanism, facile resolution of N-terminally blocked peptides from unmodified counterparts, and successful resolution on SCXHPLC of charge variants not achievable by standard RP-HPLC. The overall results were analyzed in more detail in an effort to quantitatively describe elution characteristics on this column [ 1 l] as well as comparing the SCX separation with another cation-exchange
23
matrix [ 121. General applications of sulfoethyl aspartamide SCX-HPLC peptide purification have been presented [13]. 3.2. Facile analysis of N-terminally via SCX-HPLC
blocked peptides
Figs. 2-4 present examples of the chromatographic resolution of blocked N-terminal peptides from ‘free’ N-terminal peptides as a direct comparison of SCXHPLC vs. RP-HPLC. Fig. 2 panels B and D illustrate the excellent resolution of N-terminally blocked peptides labeled II and IV, in this case acetylated, from their respective ‘free’ N-terminal peptides I (RRPYIL) and III (DRVYIHPFHLLVYS) [ 11. A positive charge is consumed by acetylation. Thus the blocked species have a net positive charge of one less than the unblocked parent molecules and so elute earlier on SCX-HPLC. For comparison, panels A and C show the corresponding RP-HPLC pattern for each blockedunblocked peptide pair. Here the elution order is reversed as the acetylated species are less polar (more hydrophobic) than the unblocked peptides. Another example of this phenomenon is shown in Fig. 3 [ 141. Pyroglutamic acid is a cyclized glutamine
II I
10.00
I 20.00
II I 30.00
. I 40.00
I 50.00
Min.
Fig. 2. RP and SCX chromatograms of N-terminal acetylated peptides. (A) RP chromatogram of peptide I = RRPYIL and peptide II = N-acetyl-RRPYIL, 140 mVolts-full scale. (B) SCX chromatogram of peptide pair I/II at 165 mVolts-full scale. (C) RP chromatogram of peptide III = DRVYIHPFHLLVYS and peptide IV = N-acetyl-DRVYIHPFHLLVYS, 555 mVolts-full scale. (D) SCX chromatogram of peptide pair IWIV at 235 mVolts-full scale. From [I] with permission.
D.L. Crimmins/Analytica Chimica Acta 352 (1997) 21-30
24
1l
pGlu
7
5
I
Q i; i
a
I
A
A
I
II
I
I RP
pGlu
l
I_-
I
,
I ____-
RP
A
L
I
I
IO
20
I
L
-
30 TIME (mid
I
40
I
50
Fig. 3. SCX and RP chromatograms of lutenizing hormone releasing factor, pEHWSYGLRPG(NHs), containing an N-terminal pyroglutamic acid (pE) and C-terminal amide (NH*). (A) SCX chromatogram of untreated peptide, 750 mVolts-full scale. (B) SCX chromatogram of PGAPase treated peptide, 375 mVolts-full scale. (C) RP chromatogram of untreated peptide, 725 mVolts-full scale. (D) RP chromatogram of PGAP’ase treated peptide, 725 mVolts-full scale. From [14] with permission.
I IO
I I 20
,L%
I
30 TIME(min)
RP I 40
I 50
j
Fig. 4. SCX and RP chromatograms of C-terminal Human Interleukin-2 Receptor peptide, QRRQRKSRRTI. (A) SCX chromatogram of stock peptide in water showing two N-terminal species, the native molecule (Gln) and the blocked derivative (pGlu), 70 mVolts-full scale. The arrow indicates the elution position of a new peak after PGAP’ase treatment of the stock peptide solution. (B) RP chromatogram of the stock peptide, 185 mVolts-full scale. From [14] with permission.
D.L. Crinunins/Analytica
residue minus one molecule of water and when present as the N-terminus of a peptide is refractory to Edman sequencing chemistry. The blocked residue can be removed with the aid of an enzyme, Pyroglutamate Aminopeptidase (PGAP’ase), yielding an N- 1 peptide with a free N-terminus. One expects then, that the deblocked peptide will elute later on SCX-HPLC than the pyroglutamyl-peptide since deblocking effectively generates an additional positive charge on the molecule. This is precisely what is observed in panels A and B of Fig. 3 [14]. Again the elution order is reversed on RP-HPLC as demonstrated in panels C and D but with less resolution under standard gradient conditions than for standard SCX-HPLC analysis. There are instances where RP-HPLC does not resolve a blocked-unblocked peptide pair but SCXHPLC does. Such an example is displayed in Fig. 4 as panels A and B [ 141. The stock C-terminal portion of Human Interleukin-2 Receptor peptide with sequence QRRQRKSRRTI apparently contains both the target peptide and a cyclized derivative as pyroglutamic acid. This was surmised from the isolation and subsequent analysis of the two SCX-HPLC peaks shown in panel A. Only the later eluting peak labeled ‘Gln’ gave Nterminal sequence following Edman degradation and treatment of the mixture with PGAP’ase resulted in loss of the peak labeled ‘pGlu’ (pyroglutamic acid) but not peak ‘Gln’. Importantly, the appearance of a new peak with greater retention time was clearly evident (indicated by an arrow) being consistent with removal of the cyclized N-terminal pyroglutamic acid residue and increase in net positive charge. Interestingly, the stock peptide solution showed no such heterogeneity when analyzed via RP-HPLC, panel B, convincingly demonstrating that SCX-HPLC can provide chromatographic separation not achievable by RP-HPLC. 3.3. Disulfide-linked following
dimers are easily isolated
SCX-HPLC
Homo- or hetero-peptide dimers covalently joined by one or more disulfide bonds are produced in a variety of ways. For instance, after cleavage of the cysteine-containing peptide from the solid-phase resin, from enzymatic/chemical cleavage of unreduced proteins, as an artifact generated during purification of cysteine-containing peptide fragments from various sources, and through synthesis to study
Chimica Acta 352 (1997) 21-30
25
for example avidity, receptor binding, enzymatic inhibition, and structural and conformational properties of dimeric vs. monomeric peptides. However produced, the homo- or hetero-dimeric species should be more positively charged than the corresponding individual monomeric constituents. For homo-dimers the charge is simply twice that of the monomeric chain and for hetero-dimers the charge is the sum of the individual peptide chains. Analytically, several methods are available to detect disulfide-linked peptides ([4], and references therein) including two-dimensional electrophoresis, pre-column sample modification with sulfhydryl reagents, post-column analysis via mass spectrometry or electrochemical detection, N-terminal Edman sequencing, and recently, in situ linkage assignment via Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry [7,15]. Generally, a combination of techniques is necessary to absolutely determine disulfide bond partners. Preparatively, three distinct chromatographic modes seem well-suited to isolate the dimer from the monomer. Size-exclusion (SE)-HPLC should separate the desired components but with attendant sample dilution and the caveat that the two components might not be resolved if the resulting threedimensional shape of the dimer is not that different from the monomer [ 161. This follows because the primary separation mechanism of SE-HPLC is governed by the shape/conformation of the analytes and strictly speaking, not the molecular weight. RP-HPLC provides a predictable elution order for the dimermonomer peptide pair because the dimer is expected to be more hydrophobic and thus retained longer on the column. Interestingly, association of two monomeric species to form a dimer can frequently mask hydrophobic residues from interaction with the column. Mechanistically, the peptide-peptide hydrophobic interaction is apparently greater than the peptidecolumn hydrophobic forces, so in this case the disulfide-linked dimer elutes earlier than the monomeric components [17]. As stated above, the net positive charge of the disulfide-linked dimer will be greater than either of the individual constituents of the monomer [4,5,7] which immediately suggests SCX-HPLC as a preparative mode of purification. As an example, two synthetic peptides, a 1Cmer ALLETYCATPAKSE with an internal position
26
D.L. Crimmins/Analytica
Chimica Actn 352 (1997) 21-30
sex
A
::I:‘1 3
Gfl
2
I
as
d
ST
10
S Y
/
ClBRP
.I
E
0*
-‘
i i
PA
c.
i-
Ak T
:
A/j
d 30 min
50
Fig. 5. HPLC purification of a Disulfide-linked Heterodipeptide. (A) SCX-HPLC of the thiol-disulfide interchange mixture of ALLETYCATPAKSE, A,-C7-Et_,, and GRGDSPC, Gt-C,. Peaks 1 through 4 were assigned from appropriate homodipeptide reactions: 1 = 2-nitro-5-thiobenzoic acid:G,-C,, 2 = 2-nitro-5-benzoic acid:A,-C,-Et4, 3 = Gt-C7:GI-C,, and 4 = At-C,-Et4:AI-C7-E14. The peak marked with a star was the desired heterodipeptide, A,CT-E,d:Gt-C,, 1750 mVolts-full scale. (B) RP-HPLC desalting of SCX purified heterodipeptide, 1750 mVolts-full scale.
cysteine and a 7-mer GRGDSPC with a C-terminal cysteine, were disulfide linked following a thiol-disulfide interchange reaction with 5,5’-dithiobis(2_nitrobenzoic acid) [4,5,7]. The chromatographic analysis is profiled in Fig. 5 with panel A representing SCXHPLC of the reaction mixture and panel B, desalting via RP-HPLC. Peaks labeled 1 and 2 in panel A are mixed-disulfide adducts with individual peptide monomers while peaks 3 and 4 are homo-dimers ascertained from independent reactions and chromatography (data not shown). The starred peak was desalted by RP-HPLC (panel B) and subjected to a battery of analytical procedures for confirmation of structure. Amino acid compositional analysis (expected/observed values) as residues per mole of dipeptide was: D (l/1.0), E (2/2.1), S (2/2.0), G (2/2.2), R (l/1.0), T (2/2.0), A (3/3.0), P (2/1.9), Y (l/0.95), C (2/net determined), L (2/l.@, and K (110.95). The correlation was good and indicates that the molar composition of the sample was consistent with the disulfide-linked peptide. Fig. 6 is a composite for the N-terminal sequence analysis of the dipeptide. As expected, the first six cycles show two residues were released corresponding to the sequence of the individual peptides. For example, at cycle 1 the two amino acids identified were A and G corresponding to the N-
Fig. 6. N-terminal Sequence Analysis of Disultide-linked Heterodipeptide, At-C7-Et4:G1-C7. S, standard mixture of phenyhhiohydantoin amino acids indicated by the usual one-letter code with X= reagent by-products. The time axis is shifted slightly to the left to avoid display overlap from cycle 1. Note, two residues were assigned in sequencing cycles 1 through 6, one residue was assigned as cystine (C,) in cycle 7, only one residue was assigned in sequencing cycles 8 through 14, and no residue was assigned in cycle 15 as expected, 40 mVolts-full scale each. Approximately 300 pmoles of sample was loaded.
terminal residue of each constituent peptide monomer. At cycle 7, a peak presumed to be diphenylthiohydantoin cystine is observed (C,) [ 181 after which cycles 8 through 14 show only residues attributable to the 14-mer peptide. Mass spectrometry gave M+ equal to 2188 vs. an expected value of 2187 along with the masses of the individual monomeric molecules [7]. There is little doubt then that the starred peak in panel A of Fig. 5 is the target hetero-dimer. Ten distinct synthetic homo-/hetero-dimers have been successfully isolated using SCX chromatography [4,5,7]. 3.4. Comparison
of SCX-HPLC
capillary electrophoresis
and free-solution
(FSCE) for peptide
analysis
Two reports have been published on the comparative analysis of SCX-HPLC vs. FSCE [19,20]. Data from each shows the elution order for mixtures of peptides is reversed on FSCE at pH 2 or 3 from that of SCX at pH 3. This is in complete accord with one’s intuition as the more positively charged molecules will migrate faster on FSCE and therefore elute earlier than
D.L. Crimmins/Analytica
Chimica Acta 352 (1997) 21-30
21
Fig. 7. SCX-HPLC and FSCE of synthetic peptides with C-terminal and methionine modifications, and truncated species. SCX-HPLC at pH 3, unprimed panels A, 1250 mVolts-full scale, B and C, 500 mVolts-full scale. FSCE at pH 3, single-primed panels, all 325 mVolts-full scale. FCSE at pH 2, double-primed panels, all 325 mVo1t.vfull scale. From [19] with permission.
less positive peptides. The electrophoretic and chromatographic techniques are thus complementary. Fig. 7 compares the SCX-HPLC profile of synthetic mixtures at pH 3 (unprimed panels) with FSCE at pH 3 (single primed panels) and pH 2 (double primed panels) [ 191. The peptide mixtures reveal another feature of the sulfoethyl aspartamide column not previously discussed, that in addition to charge-influenced separation, polarity can contribute to resolution [ 1,21-231. Panels A through C illustrate the dramatic effect of polarity, here at the C-terminus, i.e., ‘free acid’ (OH) to amide [(Nl&)], on retention properties. In all cases the more polar acid variant is retained less than the corresponding amide. Note that resolution is
better for FSCE at pH 3 (A’ through C’) compared to pH 2 (A’ through C’) but requires a longer run time. Although it is now feasible to ‘preparatively’ isolate analytes from FSCE by multiple runs using computer controlled fraction overlay it is still rather tedious and little material is actually generated. For these reasons, SCX-HPLC is preferred for the isolation of sufficient quantities of synthetic peptides. 3.5. Peptides derived by proteolysis The versatile nature of the sulfoethyl aspartamide column is further exemplified by the excellent resolution of peptide fragments generated from peptide
28
D.L. Crimmins/Analytica Chimica Acta 352 (1997) 21-30
Fig. 8. SCX and RP chromatograms following V8 protease digestion of @-Casein. (A) SCX-HPLC of digest, 140 mVolts-full scale. The numbered peaks were subjected to ammo acid analysis and N-terminal sequencing to identify the peptide fragments. The elution position of the identified peptide fragments vs. calculated net charge at pH 3 was monotonic (data not shown). (B) RP-HPLC of same digest, 250 mVolts-full scale. From [2] with permission.
mapping experiments. Proteolysis with for instance Staphylococcus aureus V8 protease, which cleaves on the C-terminal side of glutamic and aspartic acid residues, provides a predictable elution pattern for known substrates. Separation then, to a first approximation, should be a function of the number of basic residues present in the generated fragments [2,24]. Fig. 8 compares the SCX (panel A) and RP (panel B) chromatograms for a V8 digest of P-Casein [2]. The numbered peaks in the SCX profile were sequenced [2] and the experimental elution of these identified peptide fragments were consistent with the monotonic nature of charge vs. retention time reported in Fig. 1. Representative peptide mapping applications for this column include the selective isolation of C-terminal and blocked amino terminal viral proteins following proteolysis [25], isolation of two-dimensional Asp-N proteolytic peptides [26], and purification of UDPglucose dehydrogenase peptides for N-terminal sequence analysis [27]. Phosphorylation site determination for Troponin T [28], Insulin Receptor Substrate IRS-l [8], and Calmodulin [9] utilized SCX-HPLC for isolation of the labeled peptide prior to additional analysis. The retention time of the 32P-labeled peptide on this column was used to eliminate one candidate proteolytic fragment from another, thereby simplifying the
Fig. 9. HPLC purification of selected disultide-linked human von Willebrand Factor proteolytic peptides. (A) RP-HPLC of Sraphylococcus aumus VUtrypsin proteolyzed human von Willebrand Factor. Fractions were collected at 0.5 min intervals with fraction 111 (arrow) and fractions 132-137 (pooled for Asp-N digestion) taken for further analysis, 1350 mVolts-full scale. (B) SCX-HPLC of fraction 111 from A. Fractions were collected at 1.0 min intervals with fraction 37 (arrow) taken for further analysis, 90 mVolts-full scale. (C) RP-HPLC of the Asp-N digest of pooled fractions 132-137 from A. Fractions were collected at 0.5 minute intervals with fraction 110 (arrow) taken for further analysis, 1350 mVolts-full scale. (D) SCX-HPLC of fraction 110 from C. Fractions were collected at 1.0 min intervals with fraction 23 (arrow) taken for further analysis, 230 mVolts-full scale. From [7] with permission.
radiosequencing results and in combination, provided positive assignment of the phosphorylation site [8,9]. Dimeric synthetic peptides are easily identified on SCX-HPLC as shown above so it was logical to attempt to isolate disulfide-linked peptide fragments from enzymatic/chemical digests. These results were described in general for tryptic digests [29] and in particular for human complement component C3b [30] and human von Willebrand Factor [6]. The latter publication clearly showed the versatile nature of the
D.L. Crimmins/Adytica
Chimica Acta 352 (1997) 21-30
sulfoethyl aspartamide column as an indispensable aid in protein/peptide chemistry applications. Fig. 9 displays the two-dimensional chromatographic isolation of a disulfide-linked tetrapeptide (panels A and B) and a disulfide-linked tripeptide (panels C and D) [6]. It was relatively straightforward in this case to choose the appropriate fractions for further analysis because the later eluting fragments were either very basic monomeric peptides or covalently joined multimeric peptides. Edman sequencing and mass spectrometry were used to determine which possibility was the correct one. In fact, since the goal here was to assign disulfide partners in the ‘T2’ domain of human von Willebrand Factor, SCX-HPLC was used as a positive selection screening technique to generate a manageable number of candidates for subsequent analysis [6]. An interesting variation was presented for E-(y-Glutamyl)lysyl cross-Iinks in Transglutaminase-modified Human Plasminogen [31]. Again, the covalently joined dimeric peptide elutes later on SCX-HPLC due to an increase in net positive charge at pH 3. In these experiments [31] this covalent dimer actually appeared as a new SCX peak for the Transglutaminase treated protein compared to the untreated control and was thus easily isolated and structurally identified.
4. Conchsions The author has successfully used sulfoethyl aspartamide SCX columns as a versatile research tool from 1987 to the present. Many different types of samples have been analyzed during this time yet all separations in one way or another exploit differences in nominal net positive charge at pH 3 as the primary mechanism of chromatographic retention. Thus, when either consumption or generation of positive charge occurs during a chemical reaction, SCX-HPLC ought to be given prime consideration as a valuable characterization and purification technique. The interested reader is encouraged to seek other reviews on ion-exchange HPLC of peptides [11,32,33].
References [l] D.L. Crimmins, Chromatogr.
.I. Go&a, R.S. Thoma, 443 (1988) 63.
B.D. Schwartz,
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