Evaluating Immobilized Metal Affinity Chromatography for the

Agarose based immobilized metal affinity chromatography (IMAC) columns loaded with copper (II) were evaluated for the selection of histidine-containin...
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Evaluating Immobilized Metal Affinity Chromatography for the Selection of Histidine-Containing Peptides in Comparative Proteomics Diya Ren, Natalia A. Penner, Benjamin E. Slentz, Hamid Mirzaei, and Fred Regnier* Department of Chemistry, Purdue University, West Lafayette, Indiana 47907 Received February 2, 2003

Agarose based immobilized metal affinity chromatography (IMAC) columns loaded with copper (II) were evaluated for the selection of histidine-containing peptides in comparative proteomics. Recovery, binding specificity, and reproducibility were investigated with model proteins. Cu(II)-IMAC was found to be highly selective for histidine containing peptides; moreover, a low degree of nonspecific selection was observed. Acylation of the amino-terminus of peptides with either succinic anhydride, Nacetoxysuccinamide, or [3-(2,5)-dioxopyrrolidin-1-yloxycarbonyl)-propyl]-trimethylammonium (quaternary amine) reduced the number of histidine-containing peptides bound by the Cu(II)-IMAC columns. This provides an additional possibility for sample simplification in proteomic applications. The number of acylated peptides selected decreased in the order of quaternary amine > N-acetoxysuccinamide > succinic anhydride derivatization. Although the selection of N-terminally derivatized peptides is biased toward peptides that contain more than one histidine, it is not yet possible to predict selectivity. Keywords: Cu(II)-IMAC • peptide acylation • comparative proteomics

Introduction The goal in proteomics is often to identify all of the proteins in a proteome in a single study. This is facilitated by the fact that the genome of many organisms is known and modern mass spectrometers provide the molecular weight and sequence of tryptic peptides derived from proteins. Experimentally derived data is then matched with DNA and EST sequences in databases. Moreover, databases allow protein identification from only a portion of the protein sequence. When one or more peptides from a proteolytic digest are found to be complimentary to a specific gene or mRNA sequence from an EST library, it is assumed that their presence is due to expression of the entire gene or mRNA. Two strategies are currently being used to obtain the requisite experimental data needed to compare peptide molecular weight and sequence with sequences in DNA and EST databases. One is a “top-down” approach, in which intact proteins are separated, fragmented, and the fragments analyzed by MALDI/MS or ESI/MS.1,2 The top-down strategy is most widely used with 2-dimensional gel electrophoresis and, more recently, with new gas-phase approaches to protein separation and characterization. Proteomics in the “bottom-up” approach is achieved by digesting proteins first, before the fragments are separated and analyzed by mass spectrometry.3 It is widely recognized that the complexity of peptide mixtures obtained from digesting a whole proteome in bottom-up proteomics would overwhelm a mass spectrometer if they were introduced * To whom correspondence should be addressed. Phone: (765) 494-9390. Fax: (765) 494-0359. E-mail: [email protected]. 10.1021/pr034006+ CCC: $25.00

 2003 American Chemical Society

simultaneously. Preliminary fractionation before mass spectral analysis is essential. Two approaches to the analyte complexity problem in bottom-up proteomics have been explored. One is an approach described by Putnam et al. in 1983,4 where peptides from complex tryptic digests were fractionated in the first dimension by cation exchange chromatography and directly transferred to a second dimension where they were further resolved by reversed phase chromatography (RPC). A complete 2-dimensional separation was achieved in 4-12 h, depending on the number of fractions transferred from the first to the second dimension. More recently, capillary columns have been substituted for the 4.6 mm columns used by Putnam, the separation system has been coupled to a mass spectrometer, and the strategy given the name multidimensional protein identification technology (MudPIT).5,6 It is critical to note that the intent in this approach is to fractionate and analyze all of the peptides in the sample in a single analytical campaign. A second approach to bottom-up proteomics is based on efforts to decrease the complexity of tryptic peptide mixtures and reduce the number of peptides that must be examined to identify proteins. Tryptic digestion of a protein or protein subunit generally produces 30-50 peptides that range in size from a few to more than 30 amino acids. As the size of peptides increases beyond roughly 6, their sequence becomes increasing unique. In fact, their sequence can be so unique that a single peptide can be used as a signature for either the parent protein or a small number of closely related proteins. This fact has been widely exploited in signature peptide proteomics,7 global internal standard techniques (GIST),8,9 and isotopically coded Journal of Proteome Research 2003, 2, 321-329

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research articles affinity tag (ICAT)10 methods for proteomics where a representative sample of peptides from the proteome is taken that include one or more peptides from each protein. The sampling strategy in these methods is based on affinity selection of peptides that contain a low abundance amino acid. The two most frequently targeted low abundance amino acids are cysteine and histidine. Cysteine-containing peptide selection has been achieved by alkylating cysteine residues in proteins with a biotinylated reagent that allows the selection of cysteinecontaining peptides with avidin after proteolysis.3 Histidinecontaining peptides, in contrast have been selected from proteolytic digests with immobilized metal affinity chromatography (IMAC).9 Roughly 10-18% of the tryptic peptides in proteins contain either a histidine or cysteine residue, depending on the organism. In silico analyses indicate that the majority of proteins in the E. Coli (96.4%), yeast (97.0%), and human genomes (82.5%) will contain histidine residues.11 This implies that affinity selection of histidine from a tryptic digest of a proteome will greatly reduce sample complexity and still leave enough peptides for the global identification of proteins. Characterizing post-translational modifications in the signature peptide12,13 and GIST14 methods is achieved by affinity selecting modified peptides directly from digests as well. The sequence of the modified peptide serves a signature of the parent. There are a series of questions surrounding affinity selection in these bottom-up methods. One is the specificity of chemical derivatization and affinity selectors used in the method. This is an important issue because it goes to the heart of the degree to which peptides carrying the targeted amino acid are selected. If there is a high degree of specificity in selection, then the informatics component of identification will be substantially simplified by being able to assume that all peptides must contain the targeted amino acid. This would allow the searchable database to be reduced by more than 80% in the cases of cysteine- and histidine-containing peptides. Another issue is the peak capacity of the method. Separation methods that produce the largest number of fractions will be the most useful. And finally there is the matter of recovery. These issues have not been studied thoroughly in the case of either cysteine- or histidine-containing peptide selection. The work discussed below centers on histidine-containing peptides. The focus in this paper will be on IMAC selection of histidine-containing peptides. Although IMAC was introduced by Porath et al.15 in 1975 and has been widely used for the selection of histidine-containing proteins at the preparative and process scale,16 literature on IMAC selectivity with tryptic peptides is sparse. It is known that chelating stationary phases such as iminodiacetic acid (IDA) or nitrilodiacetic acid bind histidine-containing peptides reversibly when they are loaded with either Cu(II) or Ni(II).17 The number and location of histidine residues is also important in binding. Proteins with multiple histidine residues that are close together are more strongly retained than those with a single histidine or several histidine residues that are widely separated in the primary structure. Other amino acid residues such a proline, tryptophan, and R- or -amino groups can impact selectivity and retention as well.18-20 The focus of the work reported here is on the efficacy of Cu(II)-IMAC chromatography as a selector for histidinecontaining peptides. Selectivity, recovery, column longevity, reproducibility, and binding specificity were investigated with tryptic digests from model proteins varying in molecular weight 322

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Figure 1. Structures of the derivatization reagents used in this study.

and isoelectric point. The impact of derivatization agents being used for comparative proteomics was examined as well.

Experimental Section Materials and Reagents. Transferrin (human), cytochrome c (bovine), lysozyme (chicken), β-casein (bovine), bovine serum albumin (BSA), fetuin-A (bovine), TPCK-treated trypsin (bovine), copper(II) sulfate pentahydrade, urea, dithiothreitol (DTT), iodoacetic acid (IAA), N-(2-Hydrozyethyl) piperazineN′-(2-ethanesulfonic acid) (HEPES), N-tosyl-L-lysylchloromethyl ketone (TLCK), acetic acid, succinic anhydride, ethylenediaminetetra-acetic acid (EDTA) and calcium chloride were purchased from Sigma-Aldrich (St. Louis, MO). Trifluoroacetic acid (Sequenal Grade) was acquired from Pierce (Rockford, IL). Sequencing grade, modified trypsin was from Promega (Madison, WI). β-Galactosidase was purchased from Fluka (Milwaukee, WI), pyridine was from Fischer Scientific Co. (Fair Lawn, NJ), and HPLC grade acetonitrile (ACN), sodium acetate, sodium chloride, sodium phosphate dibasic heptahydrade, monobasic sodium phosphate, and phosphoric acid were obtained from Mallinckrodt Baker, Inc. (Paris, KY). Succinic2,2,3,3-d4 anhydride was from ISOTEC Inc. (Miamisburg, OH). A C18 reversed phase column (4.6 × 250 mm) was purchased from Vydac (Hesperia, CA). HiTrap Chelating HP affinity columns (0.7 × 2.5 cm) were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). A low-pressure micro-splitter valve was obtained from Upchurch Scientific (Oak Harbor, WA). Deionized water was produced by a Milli-Q Gradient A10 System from Millipore (Bedford, MA). Derivatization Reagents and Derivatization of Peptides. Three derivatization reagents were used in this work (Figure 1): succinic anhydride, the N-acetoxysuccinamide, and [3-(2,5)dioxopyrrolidin-1-yloxycarbonyl)-propyl]-trimethylamine (quarternary amine). The initial step in the synthesis of N-acetoxysuccinamide7 wastoadd3g(26.8mmol)ofN-hydroxysuccinimide to 1 g (8.9 mmol) of acetic anhydride and stir for 15 h at room temperature. White product crystals appeared gradually. Excess solvent was removed by rotary evaporation at room temperature. The white crystalline residue was washed with hexane and dried in a vacuum. The product melting point was 136138 °C. Synthesis of (3-carboxypropyl)-trimethylammonium chloride was achieved according to a method described in the literature.21 Ten g (0.097 mol) of 4-aminobutanoic acid, 48.0 g (0.497 mol) of KHCO3 and 30 mL (0.482 mol) of CH3I were stirred in

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Selection of Histidine-Containing Peptides

1L of MeOH for 24 h. The MeOH was removed by rotory evaporation and the crude residue slurried with 200 mL of CHCl3, then filtered and acidified with ∼50 mL of concentrated HCl. The H2O was rotory evaporated and the remaining residue extracted with 4 × 200 mL of anhydrous acetone, concentrated under reduced pressure, slurried with 200 mL of THF and filtered to yield a white solid (13.2 g, 75%). 1H NMR (300 MHz, D2O): δ 1.97 (m, 2H); 2.39 (t, 2H, J ) 7 Hz); 3.02 (s, 9H); 3.26 (m, 2H). 13C NMR (300 MHz, D2O): δ 18.5; 30.7 (t, JC-N ) 2 Hz); 53.7 (t, JC-N ) 3.4 Hz); 66.0 (t, JC-N ) 3.5 Hz); 177.3. The N-hydroxysuccinimide (NHS) derivative of (3-carboxypropyl)-trimethylammonium chloride was prepared according to a published method.22 13.2 g (0.073 mol) of (3-carboxypropyl)-trimethylammonium chloride, 8.4 g (0.073 mol) of NHS and 16.5 g (0.080 mol) of DCC were dissolved in CH3CN and the resulting solution refrigerated (∼-10°) for 24 h. The 1,3dicyclohexylurea formed during the reactions was removed by filtration and the CH3CN was rotary evaporated under reduced pressure. The crude residue was slurried with 200 mL of THF, filtered, and recrystallized form CH3CN/THF to yield a white solid (13.0 g, 64%). 1H NMR (300 MHz, D2O): δ 2.18 (m, 2H); 2.83 (t, 2H, J ) 7 Hz); 2.88 (s, 4H) 3.10 (s, 9H); 3.39 (m, 2H). 13C NMR (300 MHz, D2O): δ 18.5; 26.4; 28.2 (t, JC-N ) 2 Hz); 53.8 (t, JC-N ) 3.5 Hz); 65.5 (t, JC-N ) 3.5 Hz); 170.1; 174.2. A 50-fold molar excess of each derivatization reagent was added individually to a tryptic digest and the reaction was allowed to proceed for 2 h at room temperature (pH 7-8). One or two drops of pyridine were added to the succinic anhydride reaction mixture. After the reaction was finished N-hydroxylamine was added in excess and the pH was adjusted to 11-12 with sodium hydroxide to hydrolyze esters. The reaction was allowed to proceed for 10 min, then the pH was adjusted back to 7-8 with 1 to 2 drops of glacial acetic acid. Proteolysis of Proteins. Protein samples (6-9 mg/mL) were dissolved in 50 mM HEPES buffer (pH 7.8) containing 20 mM CaCl2 and 6-8 M urea. A 20× molar excess of DTT was added, and the mixture was incubated for 40 min at 37 °C. Then it was cooled to room temperature and a 40× molar excess of iodoacetic acid was added. The mixture was incubated in darkness for 2 h with subsequent addition of a 20× molar excess of L-cysteine, followed by a second incubation period of 15 min. The protein solutions were diluted 3-4-fold and trypsin was added (1/50 of total amount of protein). The solution was incubated overnight at 37 °C. Trypsin digestion was stopped by adding TLCK in a slight molar excess over that of trypsin. Cu(II)-IMAC Selection. HiTrap Chelating HP affinity columns (0.7 × 2.5 cm), which contain iminodiacetic acid (IDA) chelating groups, were used for all histidine-containing peptide selections. All chromatographic steps were performed by BioCAD 20 Micro-Analytical Workstation (Applied Biosystems, Framingham, MA). Flow rate was 1 mL/min, and detection was at 280 nm. To prepare the column for selection, first, the column was cleaned with 7 bed volumes of a 50 mM of EDTA in 0.5 M NaCl (pH 8.0), then washed with deionized H2O and equilibrated with 0.1 M NaAc (pH 4.0) in 0.5 M NaCl. Thereafter, 50 mM copper(II) sulfate was loaded to the column. Excess copper was washed from the column with 0.1 M NaAc (pH 4.0) in 0.5 M NaCl. Two elution methods were used. One approach used decreasing pH to elute histidine-containing peptides with the following four steps: the column was first equilibrated with binding buffer, which contained 20 mM phosphate (pH 7.0)

and 0.5 M NaCl; then the sample (pH 7∼8) was injected and the column was washed again with binding buffer for 10 min. The selected peptides were eluted with 0.1 M NaAc (pH 4.0) in 0.5 M NaCl. The fractions were collected, combined, and analyzed by LC-MS. Alternatively, the histidine containing peptides were also eluted with an imidazole displacer. In this approach, the column was equilibrated with 1mM imidazole in 20 mM HEPES buffer (pH 7.0, 0.2 M NaCl), then washed with 2mM imidazole in the same buffer containing 5% of acetonitrile and eluted with 50 mM imidazole in 20 mM HEPES buffer (pH 7.0, 0.2 M NaCl). LC-MS Analysis. Peptide mixtures were separated on a Vydac C18 column (4.6 × 250 mm) using an Integral MicroAnalytical Workstation (Applied Biosystems, Framingham, MA) and a mobile phase velocity of 1 mL/min with detection at 215 nm. Two mobile phases and gradient programs were used for peptide separations. Mobile phase A consisted of 0.01% trifluoroacetic acid (TFA) in deionized water. Mobile phase B contained 95% acetonitrile (ACN) with 0.01% TFA and 5% water. The elution protocol used in the “long gradient” consisted of an initial 10 min isocratic elution with 100% mobile phase A followed by a 60 min gradient ranging from 0 to 60% mobile phase B. A shorter gradient was also used consisting of an initial 10 min isocratic step followed sequentially by an 8 min gradient from 0 to 35% mobile phase B and a 14 min gradient from 35 to 60% mobile phase B. A low-pressure micro-splitter valve was used to direct 5-8% of the flow to the QSTAR workstation (Applied Biosystems, Framingham, MA) equipped with an Ion-spray source. Spectra were obtained in the positive TOF mode at a sampling rate of one spectrum per second.

Results and Discussion Elution Conditions for Cu(II)-IMAC. A trypsin digest of β-galactosidase was used in elution optimization studies. According to the literature, histidine-containing peptides are bound to Cu(II)-IMAC stationary phases with the highest efficiency between pH 7 and 8.18,19 Two general strategies have emerged for elution of bound histidine-containing peptides. One is to use a displacer such as imidazole or histidine.23,24 Another is to protonate histidine in a descending pH gradient.25-27 An alternative to this second method is to apply a single step gradient from pH 7 to 4. A sharp step gradient tends to elute histidine-containing peptides together, with little resolution. Because the objective in this work was to capture and transfer all histidine-containing peptides from the Cu(II)IMAC to the RPC column en mass, the step gradient protocol was chosen. All peaks collected after Cu-IMAC selection were combined and analyzed together by LC-MS. Mobile phase composition had a major impact on the elution of histidine-containing peptides (Table 1). Three different sets of elution conditions were examined. The mobile phase in the first contained 0.1 M sodium acetate (pH 4.0). A second mobile phase contained 0.1 M sodium acetate with 0.5 M NaCl. And the third contained 50 mM imidazole with 0.2 M NaCl in 20 mM HEPES buffer (pH 7.0). Although 10 out of the 14 β-galactosidase peptides were eluted from the Cu(II)-IMAC column with the 0.1 M sodium acetate mobile phase alone, addition of 0.5 M NaCl eluted 13 out of 14. Peptides that seem to be difficult to elute with 0.1 M sodium acetate alone were those containing multiple histidine residues. Addition of 0.5 M NaCl enhanced the elution of multiple histidine-containing peptides. The impact of 0.5 M NaCl is probably to minimize Journal of Proteome Research • Vol. 2, No. 3, 2003 323

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Table 1. Buffer, Eluting Condition, and Salt Concentration Effects on the Recovery of Histidine-Containing Peptides from an Acetate Labeled β-Galactosidase Tryptic Digest

a

peptide mass

sequence

3832.87 3423.74 3015.46 2866.38 2744.33 2522.23 2311.17 2265.20 2120.90 2006.95 1425.66 1299.62 1265.62 1252.66

AAGHYQAEAALLQCTADTLADAVLITTAHAWQHQGK HSDNELLHWMVALDGKPLASGEVPLDVAPQGK DRNHPSVIIWSLGNESGHGANHDALYR HEHHPLHGQVMDEQTMVQDILLMK NHPSVIIWSLGNESGHGANHDALYR VVQPNATAWSEAGHISAWQQWR TPHPALTEAKHQQQFFQFR DVSLLHKPTTQISDFHVATR CSHYPNHPLWYTLCDR IIFDGVNSAFHLWCNGR YHYQLVWCQK ELNYGPHQWR HQQQFFQFR LAAHPPFASWR

+

+ + + + + + +

100 mM acetate (pH 4.0, 0.5M NaCl)

+a + +a +a + + + +a + + + + +

50mM imidazole (pH 7.0, 0.2 M NaCl)

+ + + +

+

Peptides eluted late in Cu(II)-IMAC column.

ion exchange effects and in the course of doing so also potentially decrease nonspecific binding. But even with the addition of 0.5 M NaCl, it was not possible to elute the peptide HEHHPLHGQVMDEQTMVQDILLMK that contains multiple histidine residues. It is much easier to elute peptides containing multiple histidine residues from a Cu(II)-IMAC column with 50 mM imidazole. The disadvantage of the imidazole approach is that peptides containing a single histidine residue are difficult to capture. It was found that 1-2 mM of imidazole in the loading buffer was sufficient to elute weakly bound peptides containing a single histidine. Therefore, imidazole should only be used in the mobile phase when it is the objective to capture peptides with multiple histidine residues. Selectivity. Efficacy of the Cu(II)-IMAC column for selection of histidine-containing peptides was evaluated using tryptic digests of seven model proteins; cytochrome c (bovine, MW: 11.7 kDa), lysozyme (chicken, MW: 14.3 kDa), β-casein (bovine, MW: 23.6 kDa), fetuin-A (bovine, MW: 36.3 kDa), serum albumin (bovine, MW: 66.4 kDa), transferrin (human, MW: 75.2 kDa) and β-galactosidase (E. coli, MW: 116.4 kDa). The molecular weights and pI of these proteins ranged from 11 to 116 kDa and 5.1-9.6, respectively. Peptide selectivity was evaluated in a two-step procedure. The first was to separate the tryptic digest from each protein on an RPC column and identify the peptides in each fraction by mass spectral analysis. This determined which peptides from digests could be captured and eluted from an RPC column. The second step was to perform a two-dimensional chromatographic separation of the tryptic digests in which peptides captured on the Cu(II)-IMAC column were eluted and transferred to the reversed phase column where they were separated and analyzed by mass spectrometry again, as in the first step. By comparing the histidine-containing peptides captured from step one with those from step two, it was possible to determine the selectivity of the Cu(II)-IMAC column for histidine-containing peptides. Capture efficiency on the Cu(II)-IMAC was 85% or more for all the histidine-containing peptides from the model proteins. Although the Cu(II)-IMAC column captured many peptides containing a single histidine residue, the column failed to capture some. Nonspecific binding was observed in only one case. The peptide CNLLAEKQY from fetuin-A was captured nonspecifically when using the sodium acetate (pH 324

100 mM acetate (pH 4.0)

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4.0), 0.5 M NaCl mobile phase. On the basis of the fact that the tryptic digests examined in this study contained several hundred peptides, the degree of nonspecific binding observed with this IMAC column was very low. Recovery. The peptide HEHHPLHGQVMDEQTMVQDILLMK from β-galactosidase did not appear in the collected fractions eluted with the sodium acetate (pH 4.0), 0.5 M NaCl mobile phase. This is thought to be due to the close spatial proximity of histidine residues in the peptide. Peptides with adjacent histidine residues are more strongly retained on Cu(II)-IMAC columns, as opposed to those in which the histidine residues are widely separated.26,28,29 However, retention of peptides is not directly dependent on the number of histidine residues.30,31 Several cases were found where histidine-containing peptides were not found among the peptides eluted from the RPC column in step 1 but were observed in step 2, where peptides were first selected by the IMAC column and then separated on the RPC column. The only difference between these two experiments is that the mixtures applied to the RPC column were much simpler after selection of only the histidinecontaining peptides in step 2. It is likely that the explanation for the loss of peptides in the more complicated mixtures was due to suppression of ionization in the ESI-MS. Coeluting peptides sometimes suppressed the ionization of others in complex mixtures. Simplifying the mixture through IMAC selection in the second case apparently diminished the suppression of ionization. This means that suppression can have a large impact on the apparent recovery of peptides in the analysis of complex mixtures. Peptides that appear not to be recovered by mass spectrometry can actually be present but their ionization is being suppressed. It is also important to note that while analyzing the transferrin digest, a few peptides were observed that did not match any of the tryptic peptide masses in the SWISS-PROT database. Two of these “unknown” peptides at m/z 1449.65 and 1631.73 were found by MS/MS to have the sequences SVEEYANCHLAR and KPVEEYANCHLAR, respectively. These peptides have the same partial sequence as the peptide VEEYANCHLAR, which is found in the SWISS-PROT database. Transferrin in the one case had a proline residue at position 589, whereas in the other there was a serine residue. This difference in the transferrin

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Table 2. Effect of N-Terminal Derivatization on Recovery of Histidine-Containing Peptides from Transferrin peptide mass

sequence

nonderivatized

acetate

2550.27 2233.12 2070.03 1690.83 1587.76 1413.71 1318.57 1276.63 1273.65 1209.51 1196.53 1166.59 1016.49 1010.44 987.46 964.53 888.39 874.44 744.33 710.30 700.32 540.29

KPVDEYKDCHLAQWPSHTVVAR DGAGDVAFVKHSTIFENLANK EDLIWELLNQAQEHFGK DCHLAQVPSHTVVAR KPVEEYANCHLAR ELLNQAQEHFGK WCAVSEHEATK EFQLFSSPHGK HSTIFENLANK CQSFRDHMK WCALSHHER HQTVPQNTGGK KSCHTGLGR or KSCHTAVGR CALSHHEK DKEACVHK APNHAVVTR SCHTGLGR or SCHTAVGR DSAHGFLK EACVHK DSSLCK INHCR LHDR

+ + + + + + + + + + + + + + + +

+

sequence is obviously due to a single amino acid polymorphism resulting from single nucleotide polymorphism in the transferrin donors. Reproducibility. A concern with the IMAC technique is that metal ions can slowly leach from columns and impact reproducibility.18,32 This could be a substantial problem in 2-D LC when IMAC is used in the first dimension. The loss of stationary phase diminishes the phase ratio and can cause retention times to decrease. The transfer of analyte fractions into the second dimension of the LC would not be reproducible in this case. The impact of shifting retention times was minimized in these studies by applying a sharp, time-based, step gradient to cause all histidine-containing peptides to elute from the Cu(II)-IMAC column in a small volume of effluent. The fact that there were low concentrations of metal in the column effluent was not a problem in these 2-D analytical studies. Metals ions, such as Cu(II), can bind to silanol groups on RPC columns,33 but the resolution of RPC columns was not impacted when they were eluted with acidic mobile phases. A second concern was that the leaching of metal ions from the stationary phase would cause column loading capacity to diminish to unacceptable levels. The issues of both retention time reproducibility and loading capacity were examined in a study involving eight consecutive separations of a model sample on a single Cu(II)-IMAC column, without reloading Cu(II) between runs. A tryptic digest of transferrin was chosen as the model mixture. Eluents from the Cu(II)-IMAC column appeared in two, well resolved peaks (Figure 2a). The first peak was found to have 17 histidine-containing peptides, whereas the second had only 2 peptides, each carrying 2 histidine residues. The sequence of these 19 peptides can be found in the “nonderivatized” column of Table 2. Slight changes in the Cu(II)-IMAC elution profile were observed as the number of runs without reloading Cu(II) increased. Retention time of the second peak gradually decreased over the 8 sequential runs. When Cu(II) was stripped from the column with EDTA at the end of the experiment and reloaded, retention time of the second peak returned to the initial value. This strongly supports the idea that metal ion transfer and leaching from the chelating support changes retention time. When effluent containing all 19 peptides from both IMAC peaks (Figure 2a) was transferred to a reversed phase chroma-

+ + +

+

succinic anhydride

quaternary amine

+ +

+

+ +

+

+ +

+ + + + + +

Figure 2. Elution profiles of histidine-containing peptides (transferrin) from Cu-IMAC (a) and reversed phase (b) columns. Journal of Proteome Research • Vol. 2, No. 3, 2003 325

research articles tography (RPC) column, they were adsorbed at the column inlet when the mobile phase was 0.01% TFA in H2O. It is important that neither the mobile phase nor the sample contain an organic modifier. Organic modifiers can cause the elution of small, hydrophilic peptides. Even without modifier it is often the case that reverse phase columns do not capture hydrophilic peptides of 2-3 amino acids. Gradient elution of this mixture of 19 peptides from the RPC column with mobile phase ranging from 0.01% TFA in H2O to 95% acetonitrile/0.01% TFA in H2O produced the elution profile seen in Figure 2b. The numbers 1, 3, 5, and 7 in Figure 2b indicate the IMAC run from which the sample was taken. The broad peak in the early part of the chromatogram indicates that these peptides were migrating slowly through the column during application of the large volume of sample from the IMAC column. It was concluded from the transferrin digest study that the loss of loading capacity during the course of 8-10 runs would probably not be a problem (Figure 2b). The column retained sufficient loading capacity to bind sample components. But loss of Cu(II) caused a decrease in the retention time of more strongly retained peptides (Figure 2a). Although Cu leaching decreased the retention time of these peaks, the two broad groups of histidine-containing peptides were still selected. When both of these groups are collected as a single fraction, as was the case in Figure 2b, these changes in retention will not be a problem. It is only in the case of time-based selection of the second peak containing peptides with larger numbers of histidine residues that there would be a problem. For this reason, all of the histidine-containing peptides were transferred to the RPC column as a single fraction in the studies reported below. Peptide Derivatization and its Impact on Recovery and Reproducibility. Stable isotope derivatization is now widely used in comparative proteomics to code peptides according to sample origin.9,10,34,35 Quantification of change in the proteome using post-biosynthetic derivatization with stable isotope coding agents is being done via the GIST and ICAT methods. Because acylation occurs on the amino-terminus and primary amino group of lysine in the GIST method, the charge state of a peptide will be altered. There was a concern that altering the charge of a peptide could impact Cu(II)-IMAC selection. This issue was investigated by acylating peptides from tryptic digests with three different acylating agents (Figure 1); succinic anhydride, N-acetoxysuccinamide (NAS), and quaternary amine. Acylation with NAS both reduced positive charge in a peptide and made it more hydrophobic. Derivatization with succinic anhydride both eliminates a positive charge at the site of acylation and adds a negative charge. Acylation of amino groups with quaternary amine will eliminate positive change at the site of derivatization, but introduces a quaternary amine at the site simultaneously. Elution profiles of β-galactosidase tryptic digests from the Cu(II)-IMAC column using a step gradient from the initial mobile phase of 20 mM phosphate buffer (pH 7.0) containing 0.5 M NaCl to 0.1 M acetate (pH 4.0) containing 0.5 M NaCl are seen in Figure 3a. Chromatograms were found to change considerably when the tryptic digest was acylated with each of the three derivatization agents. Because the same amount of sample was applied in all cases, total peak area in this Figure gives an idea of capture selectivity. Total peak abundance decreased in the order of nonderivatized > quaternary amine > N-acetoxysuccinamide (NAS) > succinic anhydride derivatization, indicating that different amounts of peptides were 326

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Figure 3. Cu-IMAC elution profiles of labeled histidine-containing peptides from β-galactosidase (a) and transferrin (b) digests.

selected in each case. Clearly, reducing the net positive charge on the amino-terminus of peptides in the case of NAS derivatization negatively impacts the binding of histidine-containing peptides to a Cu(II)-IMAC column. Reducing positive charge and increasing the net negative charge on peptides simultaneously, as in the case of succinic anhydride derivatization, reduced binding even more. The hypothesis that decreases in peak area in Figure 3a are due to a reduction in the number of peptides captured was supported by LC/MS analysis. It was found that the number of peptides selected from β-galactosidase tryptic digests decreased from 17 for nonlabeled peptides to 12 for the case of derivatization with acetate and quaternary amine (Table 3). Only 9 peptides were found and identified in the case of succinic anhydride derivatization. Peptides with one histidine that interact weakly with the Cu(II)-IMAC stationary phase are more likely to be lost after derivatization. As expected, the

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Selection of Histidine-Containing Peptides

Table 3. Effect of N-Terminal Derivatization on Recovery of Histidine-Containing Peptides from β-Galactosidase peptide mass

sequence

nonlabeled

acetate

quaternary amine

3832.87 3423.74 3189.59 3015.46 2866.38 2744.33 2522.23 2474.19 2311.17 2265.20 2120.90 2006.95 1732.88 1507.70 1425.66 1299.62 1265.62 1252.66 1064.57 730.40

AAGHYQAEAALLQCTADTLADAVLITTAHAWQHQGK HSDNELLHWMVALDGKPLASGEVPLDVAPQGK WLSLPGETRPLILCEYAHAMGNSLGGFAK DRNHPSVIIWSLGNESGHGANHDALYR HEHHPLHGQVMDEQTMVQDILLMK NHPSVIIWSLGNESGHGANHDALYR VVQPNATAWSEAGHISAWQQWR AVVELHTADGTLIEAEACDVGFR TPHPALTEAKHQQQFFQFR DVSLLHKPTTQISDFHVATR CSHYPNHPLWYTLCDR IIFDGVNSAFHLWCNGR TPHPALTELKHQQQF YSQQQLMETSHR YHYQLVWCQK ELNYGPHQWR HQQQFFQFR LAAHPPFASWR TPHPALTAK HVATRF

+a +a + +a

+a +

+a +

+a

+a

+

+a + + + + +a +

+a +

+a +

+ +

+ +a +

+ +a +

+ + +

+ + + +

+ + + +

+ + +

a

+ + + +a + +

succinic anhydride

Peptides eluted late in Cu(II)-IMAC.

retention of peptides with larger numbers of histidine residues is impacted less by derivatization. These studies indicate that the presence of a primary amino group at the amino-terminus of peptides is very important for interaction with the immobilized metal, particularly in the case of peptides with a single histidine. The fact that binding was achieved in the presence of 0.5 M NaCl indicates that the contribution of the N-terminal amino group in single histidine peptides is more than a simple electrostatic interaction with carboxyl groups on the column. Even though this interaction was proven to be weak in comparison with histidine-Cu interaction, eliminating primary amino groups bring drastic changes in peptide behavior during selection. The free pair of electrons on the nitrogen atom of the primary amino group seems to contribute to peptide binding. The impact of acylation is much greater in the case of transferrin (Figure 3b, Table 2) where the tryptic peptides generally contain a single histidine residue. The number of peptides captured decreased sharply from 19 in the absence of acylation to 2 in the case of succinic anhydride derivatization. The retention time of quaternary amine labeled peptides is higher (Figure 3b), implying that the quaternary amino group interacts with the iminoacetate stationary phase. However, interaction with the quaternary amine is not sufficiently strong to result in nonspecific bindings of peptides lacking a histidine residue. After acylation of peptides, the number of histidine peptides selected by a Cu(II)-IMAC column can be expected to decrease in the order quaternary amine butyrate > N-acetoxysuccinamide > succinic anhydride. In addition, selection was found to depend on the proximity of histidine residues in the peptide sequence. The selection of acylated peptides was biased toward those that contain more than one histidine residue. Succinate acylation had a tendency to reject peptides with a lysine residue at their amino-terminus, but not arginine. Effect of Succinic Anhydride on Cu(II)-IMAC Column. After acylation of peptides with a large excess of succinic anhydride, residual succinic anhydride and succinate generated by hydrolysis were not removed before Cu(II)-IMAC fractionation. Severe leaching of Cu from the column was noticed with these

Figure 4. Cu-IMAC elution profiles of nonlabeled histidinecontaining peptides from transferrin before and after three subsequent selections of succinic anhydride labeled sample.

samples. The blue color of the column gradually faded quickly during column use, especially at the inlet. Two samples of transferrin peptides were examined (Figure 4). One was analyzed with an IMAC column on which Cu(II) had been freshly loaded. The second chromatogram was obtained with the same column after three separations of the succinic anhydride derivatized transferrin peptide sample. Retention times and peak areas were seen to decrease (Figure 4) even faster than those noted in Figure 2a. Again, this is interpreted to be the result of Cu(II) loss from the column. Because succinate is known to chelate Cu(II), it apparently competes with IDA on the surface of the sorbent for Cu(II). When derivatizing with succinate, it would be prudent to either remove unreacted succinate before Cu(II)-IMAC or reload the column with Cu(II) before each separation. Tuning Cu(II)-IMAC Selection. Amino-acylation of peptides is seen in Table 4 to give additional opportunity for sample simplification beyond that obtained with native tryptic peptides. For example, only 7% of peptides from transferrin are Journal of Proteome Research • Vol. 2, No. 3, 2003 327

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Ren et al.

providing 77% coverage. When results from a double histidine selection method described here and a histidine/cysteine selection method are combined, in silico analysis shows that coverage approaches 100%. This would be a rapid, simple alternative to the MudPIT method.

Conclusions

Figure 5. Reversed-phase separation of a tryptically digested Yeast extract before and after selection, with and without acetate derivatization. Table 4. Sample Simplification (%) for Transferrin and β-Galactosidase Digests with and without N-Terminal Derivatization after Cu(II)-IMAC Selection proteins

nonlabeled

acetate

succinic anhydride

quaternary amine

β-galactosidase transferrin

27 31

19 7

14 3

19 15

selected after acetate derivatization, versus 31% if derivatization was not performed. This means that acylation provides the possibility for “tuning” selection according to experimental goals. Figure 5 shows the reversed-phase separation of a tryptic digested Yeast extract before and after selection, with and without derivatization. It is clear that sample complexity, number of peaks, and UV absorbance decreases after selection. This effect is even greater when the acetate labeled digest was used for selection. According to in silico analyses, a tryptic digest of the E. coli proteome would contain 150 000 peptides if all possible proteins were expressed. Considering only tryptic peptides that contain histidine, roughly 15% of the 150 000 tryptic peptides would contain one or more histidine residues and represent greater than 90% of all proteins in the proteome. The fraction of tryptic peptides that would contain two or more histidine residues drops to approximately 3%, or roughly 5000 peptides. It is interesting that although the number of peptides drops 5-fold in going from all histidine-containing peptides to those that have two or more histidine residues, coverage of the proteome only drops from 90% to roughly 65-70%. This means that Cu(II)-IMAC selection methods that select tryptic peptides with two or more histidine-containing residues should theoretically reduce sample complexity by 97%, while still giving 65-70% coverage. When the objective is to obtain 100% coverage of the proteome and still use drastically simplified mixtures, double selection might be a good solution. Using a histidine and cysteine double selection method roughly 6% of the peptides in the human proteome would be sampled while 328

Journal of Proteome Research • Vol. 2, No. 3, 2003

Cu(II)-IMAC was evaluated as a method for sample simplification in comparative proteomics. It is concluded that when Cu(II)-IMAC columns are used as the first column in a twodimensional liquid chromatographic fractionation of tryptic peptides they provide high selectivity in the selection of histidine containing peptides. Column selectivity can be varied though the use of either the type of mobile phase displacing agent, salt concentration in the mobile phase, or acylation of amino groups in peptides. This method was proven to provide reproducible and accurate results with a low degree of nonspecific selection. The number of peptides selected decreases when peptides are amino acylated. Peptides containing two or more histidine residues are selected predominantly after acylation. Cu(II)-IMAC selection can thus be a valuable tool in proteomic studies.

Acknowledgment. The authors greatly acknowledge financial support from Grants 5R01 GM 5996-04. Dr. Miroslav Sedlak (Laboratory of Renewable Resources Engineering, Potter Engineering Center, Purdue University, West Lafayette, IN) is acknowledged for providing Yeast samples References (1) Kelleher, N. L.; Lin, H. Y.; Valaskovic, G. A.; Aaserud, D. J.; Fridriksson, E. K.; McLafferty, F. W. J. Am. Chem. Soc. 1999, 121, 806-812. (2) Reid, G. E.; McLuckey, S. A. J. Mass Spectrom. 2002, 37, 663675. (3) Aebersold, R.; Goodlett, D. R. Chem. Rev. 2001, 101, 269-295. (4) Takahashi, N.; Takahashi, Y.; Putanm, F. W J. Chromatogr. A. 1983, 266, 511-522. (5) Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R. Nat. Biotech. 1999, 17, 676-682. (6) Washburn, M. P.; Wolters, D.; Yates, J. R. Nat. Biotech. 2001, 19, 242-247. (7) Geng, M.; Ji, J.; Regnier, F. J. Chromatogr. A. 2000, 870, 295-313. (8) Wang, S.; Regnier, F. E. J. Chromatogr. A. 2001, 924, 345-357. (9) Ji, J.; Chakraborty, A.; Geng, M.; Zhang, S.; Amini, A.; Bina, M.; Regnier, F. J. Chromatogr. B. 2000, 745, 197-210. (10) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat. Biotech. 1999, 17, 994-999. (11) Wang, S.; Zhang, X.; Regnier, F. E. J. Chromatogr. A. 2002, 949, 153-162. (12) Geng, M.; Zhang, X.; Bina, M.; Regnier, F. J. Chromatogr. B. 2001, 752, 293-306. (13) Riggs, L.; Sioma, C.; Regnier, F. J. Chromatogr. A. 2001, 924, 359368. (14) Regnier, F. E.; Riggs, L.; Zhang, R.; Xiong, L.; Liu, P.; Chakraborty, A.; Seeley, E.; Sioma, C.; Thompson, R. J. Mass Spectrom. 2002, 37, 133-145. (15) Porath, J.; Carlsson, J.; Olsson, I.; Belfrage, G. Nature 1975, 258, 598-599. (16) Chicz, R. M.; Regnier, F. E. Anal. Chem. 1989, 61, 1742-1749. (17) Chaga, G. S. J. Biochem. Biophys. Methods 2001, 49, 313-334. (18) Hansen, P.; Lindeberg, G.; Andersson, L. J. Chromatogr. 1992, 627, 125-135. (19) Andersson, L.; Sulkowski, E. J. Chromatogr. 1992, 604, 13-17. (20) Yip, T. T.; Nakagawa, Y.; Porath, J. Anal. Biochem. 1989, 183, 159171. (21) Chen, F. C. M.; Benoiton, N. L. Can. J. Chem. 1976, 54, 33103311. (22) Anderson, G. W. Patent; Am. Cyanamid; FR 1406785; 1965. (23) Kastner, M.; Neubert, D. J. Chromatogr. 1991, 587, 43-54. (24) Sulkowski, E. J. Mol. Recognit. 1996, 9, 389-393. (25) Porath, J. Protein Expression Purif. 1992, 3, 263-281.

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