Studies of Histidine As a Suitable Isoelectric Buffer for Tryptic

Sep 6, 2011 - The use of histidine as a protein digestion buffer followed by isoelectric trapping separations using “membrane separated wells for is...
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Studies of Histidine As a Suitable Isoelectric Buffer for Tryptic Digestion and Isoelectric Trapping Fractionation Followed by Capillary Electrophoresis Mass Spectrometry for Proteomic Analysis Stephanie M. Cologna, Brad J. Williams, William K. Russell, Pei-Jing Pai, Gyula Vigh, and David H. Russell* Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States

bS Supporting Information ABSTRACT: The use of histidine as a protein digestion buffer followed by isoelectric trapping separations using “membrane separated wells for isoelectric focusing and trapping” (MSWIFT) and mass spectrometry (MS) analysis is described. Tryptic digestion of bovine serum albumin (BSA) performed in histidine buffered solutions yields similar amino acid sequence coverage values to those obtained using ammonium bicarbonate buffer. Time course studies suggest that histidine buffers provide faster migration of peptides from the loading compartment compared to digestions prepared in ammonium bicarbonate due to differences in conductivities of the two buffers. In addition, this sample preparation method and MSWIFT separations have been coupled with capillary electrophoresis (CE) and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) as an alternative separation approach for proteomic studies. Tryptic peptides of ribosomal proteins in histidine are fractionated using MSWIFT followed by CE MALDI-MS, which further illustrates the ability to couple fractions from a pI based separation device to CE MS. Specifically, two-dimensional CE MS plots provide a direct correlation between the numbers of basic residues within the peptide sequence displayed in charge-state trend lines. Combining MSWIFT and CE MS provides added information regarding peptide sequence, specifically pI and in-solution charge state. Post-translational modifications can also be identified using this method.

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soelectric point (pI) based separation techniques and specifically isoelectric trapping (IET) are useful for the separation of peptides and proteins in the absence of carrier ampholytes. In IET, buffering membranes are used to create a stepwise pH gradient in a compartmentalized setup referred to as a multicompartmental electrolyzer (MCE).1 4 During electrophoresis, ampholytes migrate until reaching a compartment in which the pI value falls between the pH values of the buffering membranes. Thus, the component is “trapped” in a compartment based on pI. Several analytical-scale MCEs have been introduced for proteomic applications2,5 8 including our in-house device termed “membrane separated wells for isoelectric focusing and trapping” (MSWIFT).9 We have demonstrated that MSWIFT is an efficient prefractionation device used to separate peptides followed by MS and liquid chromatography tandem mass spectrometry (LC MS/MS) for bottom-up proteomic studies.10 One advantage of using the MSWIFT device is the ability to apply higher electrical power loads (e.g., watts) compared to the power load capabilities of commercially available analytical devices. The benefit of applying a high electric field is a reduction in separation time; however, regardless of the device, the conductivity of the sample can inherently affect the results of the separation. Recently, Mann and co-workers11 described a proteomics approach to analyze yeast peptides using the OFFGEL Fractionator (Agilent Technologies). The authors note that “salt r 2011 American Chemical Society

concentration is critical” and that “buffering conditions with 50 mM ammonium bicarbonate resulted in failure of the isoelectric focusing (IEF).” These observations could be a direct correlation to the conductivity of the sample considering successful fractionation was achieved using 4-(2-hydroxyethyl)-1piperazineethane sulfonic acid (HEPES) buffer, which has a significantly lower conductivity. Therefore, samples with low conductivities would separate more rapidly than those prepared in higher conductivity buffers. The concept of using low conductivity buffers has been described for capillary zone electrophoresis,12 14 including histidine,15,16 carrier ampholytes,17 or isoelectric buffers for IET separations.18 Capillary electrophoresis (CE) coupled with mass spectrometry (CE MS) has emerged as a powerful tool for proteomics19 and biomarker discovery.20 22 We have recently shown the added utility of charge-state specific trends revealed in two-dimensional plots of log (μeff) versus log (MW) for CE MS data of proteolytic protein digests.23 These chargespecific trends provide a direct correlation to the number of basic residues in each peptide along a trendline. Therefore, with the use of two-dimensional plots obtained from CE MS Received: May 16, 2011 Accepted: September 6, 2011 Published: September 06, 2011 8108

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Analytical Chemistry experiments, the in-solution charge state can be used as an indication of charge state altering modifications or as added information to assist in peptide and/or protein identification. In the current work, we investigate the use of histidine as a tryptic digestion buffer for MS-based proteomic studies. Furthermore, separations of tryptic peptides prepared in ammonium bicarbonate buffer using the MSWIFT device is compared to results obtained using a lower conductivity buffer, specifically histidine. For proteomic studies, we use the histidine sample preparation method, MSWIFT fractionation, and CE MS/MS analysis of ribosomal proteins. With the MSWIFT fraction coupled with CE MS, estimated pI values and in-solution charge states corresponding to the number of basic residues can be obtained from peptides providing an additional dimension of information to assist in assignment.

’ EXPERIMENTAL METHODS Chemicals. Chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and used as received unless otherwise noted. HPLC grade methanol and isopropanol (IPA) were obtained from EMD Chemicals Inc. (Gibbstown, NJ). Formic acid (99%) was purchased from Acros Organics (Morris Plains, NJ). Trypsin (sequencing grade) was purchased from Promega (Madison, WI). All experiments were performed with 18 MΩ water (ddH2O) purified using a purification unit from Barnstead International (Dubuque, IA). IET Using the MSWIFT Device. Details regarding the design and assembly of the MSWIFT device have been previously described.9,24 The main housing of the MSWIFT device was built with polycarbonate and equipped with an aluminum heat sink. Alumina separation compartments were assembled serially to obtain the desired number of separation wells and filled with up to 200 μL of sample solution. Poly(vinyl-alcohol) based buffering membranes with tunable pH values were synthesized in-house.25 27 The anode compartment contained 3 mM methanesulfonic acid, whereas 3 mM sodium hydroxide was used as the cathode buffer. Typically, separations were carried out at 5 W constant power. Theoretical pI values were determined using the compute pI/MW tool from ExPASy.28 Theoretical conductivity and buffering capacity values were obtained using Peakmaster, version 5.2.29 Solution conductivity measurements were performed using a model 145A conductivity meter (Thermo Orion, Waltham, MA) equipped with a MI-900 series conductivity electrode (Microelectrodes Inc., Bedford, NH). Proteolytic Digestion and Sample Preparation. In total, 10 μg of bovine serum albumin was dissolved in 10 μL of either 5 mM histidine or 25 mM ammonium bicarbonate. The protein was denatured thermally at 90 °C for 10 mins and allowed to cool to room temperature. Trypsin was added at a ratio of 1:50 (w/w), and the solutions were incubated at 37 °C overnight. A mixture containing 10 μg of each of the following five proteins, bovine serum albumin, bovine apo-transferrin, bovine αs1-casein, bovine ribonuclease A, and horse cytochrome C, was dissolved in 200 μL of either 10 mM histidine or 25 mM ammonium bicarbonate. The protein mixture was reduced and denatured with 5 mM tris(2-carboxyethyl)phosphine at 60 °C for 1 h. Alkylation was performed by the addition of 2 mM methyl methanethiosulfonate and incubated at room temperature for 10 mins. Trypsin was added at a ratio of 1:50 (w/w) enzyme/protein, and the solution was incubated at 37 °C overnight. For time point analyses, the digested protein solution was

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loaded in the pH 6.8 7.6 well. A 10 μL pipet was used to mix the contents in each well, and a 10 μL aliquot from each well of the MSWIFT device was removed at 1, 3, 5, 10, 20, 30, and 45 min. From the 10 μL aliquot removed, 1 μL was mixed with CHCA and spotted for MALDI-MS analysis. The pH values of the buffering membranes used in this separation were 2.9, 4.2, 5.4, 6.8, 7.6, 9.5, and 11. 70S ribosomes were isolated from E. coli as previously described.30 Ribosomal RNA was removed by acid precipitation (1% (v/v) TFA) followed by centrifugation. A total of 100 μg of soluble proteins were precipitated using 6 volumes of excess cold acetone followed by centrifugation at 14 000 rpm  10 min. The protein pellet was resuspended in 5 mM histidine buffer followed by trypsin digestion (1:100 (w/w) enzyme/protein ratio) at 37 °C overnight. Following the initial digestion step, the protein solution was thermally denatured at 60 °C for 1 h then allowed to digest for an additional 3 h with a subsequent addition of trypsin as previously described.31 The pH values of the buffering membranes used for this separation were 2.9, 4.7, 5.4, 6.8, 7.5, 8.4, and 11.0. The sample was loaded in the well bracketed by pH 7.5 and 8.4 buffering membranes. Resulting MSWIFT fractions were concentrated 10-fold and resuspended in 0.5 mM histidine buffer to prepare for CE MS analysis. For comparison, 1 μL from each well was diluted 10-fold using CHCA and spotted for MALDI-MS and MS/MS analysis. CE MALDI. A layer of 5 mg mL 1 α-cyano-4-hydroxycinnamic acid (CHCA) was applied to the MALDI target prior to the CE separations using an x y z translation stage (ProBot, LC Packings, Sunnyvale, CA). CHCA was dissolved in 12:7:1 MeOH/ddH20/IPA (v/v/v) with 10 mM dihydrogen ammonium phosphate and doped with 50 fmol μL 1 bradykinin fragment (2-9) (PPGFSPFR) and 150 fmol μL 1 ACTH fragment (18-39) (RPVKVYPNGAEDESAEAFPLEF) as internal calibrants. The CHCA matrix solution was infused through a fused-silica capillary at 1.0 μL min 1 with a 5 s spotting interval to prepare a 30 row by 30 column array (900 total spots). MALDI plates were used for CE fraction collection using a sheath liquid composed of 50:50 MeOH/250 mM FA (0.5 μL min 1 flow rate) as the overlayer. CE separations were carried out on a 70 cm (50 μm i.d., 360 μm o.d.) fused-silica capillary (Polymicro Technologies, Phoenix, AZ) with 200 nm UV detection at 50 cm on a homebuilt apparatus. The cathodic end-capillary tip was interfaced to a ProBot (LC Packings, Sunnyvalve, CA) as previously described.23 The CE capillary was coated with a neutral linear polyacrylamide dynamic coating (Ultratrol LN, Target Discovery, Palo Alto, CA). The background electrolyte consisted of 250 mM formic acid at pH 2.20. Sample stacking injections were performed by injecting at 2.0 psi for 20 s corresponding to 65 nL injection volumes. Separations were performed at a potential of +25 kV, using a 30 kV high-voltage power supply (Gamma High-Voltage Research, Ormond Beach, FL). UV electropherograms were collected using a home-built virtual instrument interface in the LabView 7.1 (National Instruments, Austin, TX) environment. CE fractions were collected using the ProBot with 10 s spotting intervals. MALDI-MS and MALDI-MS/MS. All MALDI-MS experiments were performed using a 4700 Proteomics Analyzer MALDI-timeof-flight/time-of-flight (TOF/TOF) mass spectrometer (Applied Biosystems, Foster City, CA). For direct MS analysis, samples were mixed 1:1 (v/v) with 5 mg mL 1 CHCA. The MS data were acquired using the reflectron detector in positive ion mode (700 4500 Da) with external calibration. Collision induced 8109

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Figure 1. MALDI mass spectrum of a tryptic digestion of bovine serum albumin (10 μg) in (A) 25 mM ammonium bicarbonate and (B) 5 mM histidine. Amino acid sequence overages were 52% for the ammonium bicarbonate buffer digestion compared to 56% for the histidine buffer digestion. Peaks denoted with asterisks correspond to peptides from bovine serum albumin confirmed by peptide mass mapping.

dissociation tandem MS spectra were acquired using air at the medium pressure setting and at 1 kV of collision energy. All MS and MS/MS data were searched against the Swiss-Prot protein sequence database using the GPS Explorer (Applied Biosystems) software and an in-house license of MASCOT (version 2.1). Database searching parameters were as follows, taxonomy, Metazoa; precursor mass tolerance, 50 ppm; enzyme, trypsin; missed cleavages, one; and variable modifications, MMTS (C), oxidation (M). Database searches performed for ribosome data included the same parameters with the exception of the taxonomy being Escherichia coli. Protein identifications were considered at the 99.99% confidence interval. Ribosomal protein identifications obtained from MSWIFT-CE MALDI-MS/MS analysis are included in the Supporting Information (Table S1).

’ RESULTS AND DISCUSSION The majority of mass spectrometry-based proteomics have utilized trypsin for proteolytic digestion for several reasons including (i) site-specific cleavage at the basic residues arginine and lysine, which assists ionization and database searching, (ii) typical peptide molecular weights distributions between 700 and 2000 Da, and (iii) trypsin has high activity at physiological pH. While tryptic digestion is not sensitive to the chemical composition of the buffer, buffers such as ammonium bicarbonate are preferred since they do not adversely affect downstream analysis (e.g., mass spectrometry). On the other hand, excessive Joule heating caused by high conductivity of buffers such as ammonium bicarbonate preclude the use of high electric field strengths which are necessary for efficient electrophoretic separations.12

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Figure 2. MALDI mass spectra taken from a time course study using a mixture of five proteins digested with trypsin. The spectra below are taken from an aliquot of the solution in the separation well bracketed by pH 6.8 and 7.6 buffering membranes after 3 min of isoelectric trapping separation in the ammonium bicarbonate buffer (A) and the histidine buffer (B). Peaks are labeled with their calculated pI values.

Histidine is an ideal, cost-effective, isoelectric buffer for tryptic digestion with a solution pH value of ∼7.5 at low millimolar concentrations and its low conductivity facilitates fast electrophoretic separations. On the basis of these points, the goals of this work are threefold: (i) to investigate the utility of histidine as a sample and digestion buffer, (ii) determine if the histidine buffer indeed reduces sample separation times as expected due to the low conductivity of the solution, and (iii) implement the histidine buffering sample preparation method for MSWIFT-CE MS analysis of biological samples. Figure 1 contains representative MALDI mass spectra for the tryptic digestion of bovine serum albumin performed in either histidine or ammonium bicarbonate buffer. Ion signals labeled with asterisks denote tryptic peptides from albumin that have been assigned by accurate mass measurement/peptide mass mapping. The amino acid sequence coverage obtained from these digests are 52% in ammonium bicarbonate and 56% in histidine buffer indicating that histidine buffer is compatible with trypsin digestion of proteins. To ensure that enzyme activity is not hindered owing to the lower pH value of the histidine buffer system, we analyzed the tryptic digestion of albumin in a timecourse study in both ammonium bicarbonate and histidine buffers. An aliquot of the digestion mixture at 0, 30, 60, 180, and 360 min following trypsin addition was taken for MALDIMS analysis, and the data are provided in the Supporting Information (Figure S3). Though some spectral differences can be observed, specifically with respect to relative ion abundance, peptide mass mapping results from the 1 h time point result in 38% amino acid sequence coverage in the ammonium bicarbonate buffer compared to 32% sequence coverage for the histidine preparation. For separations using MSWIFT, the conductivity of the solution in a particular well dictates the effective field strength for that well, i.e., high conductivity will result in low field strengths.12 8110

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Figure 3. MALDI mass spectrum taken from a time course study using a mixture of five proteins digested with trypsin. The spectra below are taken from an aliquot of the solution in the separation well bracketed by pH 5.4 and 6.8 buffering membranes after 3 min of isoelectric trapping separation of the sample loaded in ammonium bicarbonate buffer (A) and histidine buffer (B). Peaks are labeled with their calculated pI values.

That is, separations performed in higher conductivity buffers, i.e., ammonium bicarbonate, are less efficient and require longer separation times when compared to low conductivity buffers such as histidine. To illustrate this point, we performed a time course study of a five-protein mixture digest using the MSWIFT device. The tryptic digest of five standard proteins was loaded in the fourth separation well of the MSWIFT device and aliquots were taken at various time points and analyzed using MALDIMS. Figure 2A contains the MALDI mass spectrum of analytes from the fourth well (bracketed by pH 6.8 and 7.6 buffering membranes) after 3 min of separation of the digest prepared in ammonium bicarbonate buffer. The mass spectra contain several ion signals corresponding to tryptic peptides from the five proteins. The theoretical pI values are provided for each of the assigned peptides. The complexity of the mass spectrum taken after 3 mins of separation in histidine buffer (Figure 2B) is reduced compared to spectra from a comparable sample taken after 3 mins of separation in ammonium bicarbonate buffer (Figure 2A). The mass spectral data suggest that separation in histidine occurs at a much faster rate as indicated by the reduced number of low pI peptides remaining in the well following 3 min of separation. MALDI mass spectra are also provided as Supporting Information for the digestion mixture prior to separation (Figure S1) and for the loading fraction (pH 6.8 7.6) at 3, 10, and 30 min of separation in both buffer systems (Figure S2 in the Supporting Information). To test whether peptides are trapped in the proper fraction, the MALDI mass spectra of aliquots taken from the adjacent well (pH 5.4 6.8) at the 3 min time point are also provided (Figure 3); the spectrum shown in Figure 3A was taken from the ammonium bicarbonate buffer experiment, and the spectrum shown in Figure 3B was taken from the histidine buffer experiment. Note that a larger number of peptide ion signals are

Figure 4. Plot of molecular weight versus theoretical pI value (A) and plot of theoretical pI value versus in solution charge at pH 2.2 (B) for an in silico tryptic digestion of bovine serum albumin from molecular weight 500 4000 Da allowing for one missed cleavage.

obtained for the histidine separation (Figure 3B) suggesting that more peptides are transported out of the loading well to the more acidic wells. Additionally, the pI values of the tryptic peptides observed in the ammonium bicarbonate experiment (Figure 3A) are lower, corresponding to more acidic peptides, than those observed in the histidine experiment (Figure 3B) for the same fraction, meaning that while the very acidic peptides are being migrated, it is much slower in ammonium bicarbonate compared to histidine. Conversely, the spectrum acquired from the histidine experiment contain several peptides that belong in that specific well and few acidic peptides are present suggesting that the majority of the acidic peptides have already moved to the proper fraction. One peptide ion signal that was of interest to us was observed at m/z 2249.00. This peptide was observed in the MALDI spectrum of the digests performed in both buffers as well as in Figure 3B. The peptide is assigned as 10 IFVQKCAQCHTVEK23 with the heme attached from horse cytochrome c, and this assignment is supported by accurate mass measurement, isotope distribution, and tandem MS data (not shown). The heme is known to covalently bind at amino acid residues Cys15 and Cys18 which are included within this tryptic peptide fragment.32 Although the theoretical pI value for this peptide is 8.1, the binding of the heme molecule may lead to a change in the isoelectric point owing to the acidic nature of the heme group.33 8111

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Figure 5. (A) Typical 2D plot of log(μeff) versus log(MW) for CE MS data obtained from MSWIFT fraction 1 (pH range 2.9 4.7). (B) Table summarizing proteolytic peptides labeled along each charge-state trend. The peptide amino acid sequence, ribosomal protein, observed m/z values, migration time (tm), theoretical pI, measured charge state (q), log (μeff), and log(MW) are given for each peptide.

The results described above suggest that histidine is an efficient tryptic digestion buffer, a result that has significant implications for electrophoretic separations. Specifically, in the analysis of complex mixtures, viz., whole cell lysates, which contain hundreds or even thousands of proteins, there are real advantages for employing multiple separation techniques to fully characterize the proteome, especially for detection of lowabundance proteins which are typically of great interest. The ability to perform multiple separation steps is an inherent advantage for the MSWIFT sample fractionation approach. For example, the orthogonality afforded by using an IEF device similar to MSWIFT followed by CE is estimated to be ∼47%, which compares favorably with the commonly used SCX-RP LC coupling with an estimated orthogonality of 53%.34 Figure 4 contains a plot of molecular weight vs theoretical pI (Figure 4A) for an in silico digest of bovine serum albumin considering one missed cleavage. This plot illustrates the distribution of pI values across the digest: the majority of tryptic peptides possess acidic pI values similar to findings obtained in our laboratory (data not shown). Combining MSWIFT with CE MALDI MS is valuable owing to the added dimension of information, specifically insolution charge state,23 that can be obtained. The solution charge state of peptides can be determined based on charge-state trend lines that are observed in plots of log (μeff) versus log (MW). The resulting charge-state trend lines can be useful as validation criteria in peptide identification. Figure 4B contains a plot of theoretical pI versus predicted in-solution charge state for a

tryptic digest of bovine serum albumin at pH 2.2. The distribution of theoretical pI values over charge states of 1+ through 5+ indicates that coupling these two methods is advantageous for proteomic analysis. In addition to using estimated pI information obtained from MSWIFT fractionation, including charge state information from CE MS can result in increased confidence of peptide assignment. To further illustrate the advantages of the histidine buffer system with MSWIFT-CE MS, we examined a more complex biological system, the 70S ribosome from E. coli. Ribosomal proteins were subjected to tryptic digestion using the histidine buffer conditions previously described. The use of histidine as a low conductivity, isoelectric buffer for MSWIFT fractionation is also advantageous for sample-stacking in CE.35,36 CE sample stacking methods allow for increased sample loading which in turn assists with increasing peptide and protein identifications for proteomic studies. The MALDI-MS/MS analysis of the ribosomal protein digest without sample separation yields identification of 12 ribosomal proteins as compared to 20 IDs obtained by using MSWIFT-MALDI-MS/MS. CE MALDI-MS/MS of the ribosomal protein digest alone resulted in the identification of 27 ribosomal proteins, and coupling the two techniques, i.e., MSWIFT-CE MS/MS yields 32 ribosomal protein IDs, clearly showing an increase from single fractionation or no fractionation (Table S1 in the Supporting Information). The results presented above clearly demonstrated that pIbased fractionation methods are compatible with CE separation 8112

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Analytical Chemistry in the second dimension followed by MALDI-MS/MS analysis. Similar work performed by Girault et al.34 shows that coupling these two electrophoretic methods can be an alternative to LC MS analysis; however, coupling of electrophoretic techniques adds additional information. For example, Figure 5A contains the two-dimensional plot obtained from CE MALDI analysis of the pH 2.9 4.7 fraction of the MSWIFT. Note that these plots contain distinct trend lines corresponding to the 1+, 2+, and 3+ ion charge states. Analysis of the trend lines can be used to enhance confidence limits for peptide IDs. For example, the data point corresponding to 20QYDINEAIALLK31 (labeled D on the 2+ trend line in the plot from the ribosomal protein L1 observed at m/z 1390.73) has a theoretical pI value of 4.37 and a predicted charge of 2+ owing to protonation of the N-terminus and the C-terminal lysine residue. This result illustrates that peptides that are similar in both mass and pI can be further separated on the basis of differences in charge states. For example, the ribosomal L24 tryptic peptide 7RDDEVIVLTGK17 (m/z 1244.68, pI 4.56, 58 MS/MS ion score) denoted as A falls on the 3+ trend line, while 110ALEEAGAEVEVK121 (m/z 1244.62, pI 4.09, 63 MS/MS ion score) from the L7/L12 ribosomal complex is located on the 2+ trend line. Separation of these two peptides (m/z values differ by 0.06 Da) requires >22 000 mass resolution for complete resolution. Note that only MSWIFT fraction 1 contains 1+ charge state peptides, which could correspond to peptides with a charge altering modification (e.g., sulfation, phosphorylation, or acetylation). The 1+ charge state peptides 79FISIEAE85 (m/z 808.37, pI 4.24) and 135SM(ox)GLVVED142 (m/z 865.38, pI 4.03) correspond to the C-terminal peptides from the ribosomal proteins L27 and L11, respectively. Overall, the added MSWIFT-CE MS separation scheme provides an accurate mass measurement, estimated pI range, and the ion charge state information, which increases the confidence of the peptide and therefore protein identification.

’ CONCLUSIONS Here, we show the use of a low conductivity buffer (e.g., histidine) for proteolytic digestion using trypsin followed by electrophoretic separations. Histidine is an excellent choice for a sample buffer to carry out tryptic digestion, and improved electrophoretic efficiency is observed using pI-based techniques (via MSWIFT) as well as by stacking CE MS analysis owing to its low conductivity. These experiments highlight the importance of buffer selection and how separations can improve when considering buffer composition. Moreover, following proteolytic digestion performed in histidine, electrophoretic based separation methods can be easily performed owing to the low conductivity of the buffer. Hence, the low conductivity of histidine allows sample stacking injections to be performed directly after IET, which improves the CE sample loading capacity. Peptide information that can be obtained from both electrophoretic methods can further be used to increase peptide assignment confidence and coupling also increases the number of protein identifications that can be made by extending the overall peak capacity. In this work we have successfully used histidine as a buffer for tryptic digestion of a single protein and for complex protein mixtures. Amino acid sequence coverage is not compromised suggesting that histidine is comparable to ammonium bicarbonate as a digestion matrix. Additionally, we performed pI based separations using the MSWIFT device followed by MALDI-MS/

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MS analysis to compare the separation efficiency between the histidine and ammonium bicarbonate buffer. Histidine buffer is an excellent choice for rapid separations and does not sacrifice the quality of the separation. Following the MSWIFT separation, collected fractions are further separated using CE and analyzed using MALDI-MS/MS. Peptides with similar pI values but different in-solution charge states (owing to the number of basic residues) are distinctly identified in two-dimensional CE MS plots of log(μeff) vs log (MW). By incorporation of these two separation techniques, estimated pI and charge state values are obtained to further aid in protein identification.

’ ASSOCIATED CONTENT

bS

Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Fax: (979) 845-9485. Phone: (979) 845-3345.

’ ACKNOWLEDGMENT This work has been supported by grants from the U.S. Department of Energy, Division of Chemical Sciences, Basic Energy Sciences (Grant BES DE-FG02-04ER15520), the Gradipore Chair in Separation Science at Texas A&M University, and the Office of the Vice President for Research, Texas A&M University. ’ REFERENCES (1) Martin, A. J. P.; Hampson, F. J. Chromatogr. 1978, 159, 101–110. (2) Righetti, P. G.; Barzaghi, B.; Luzzana, M.; Manfredi, G.; Faupel, M. J. Biochem. Biophys. Methods 1987, 15, 189–198. (3) Faupel, M.; Barzaghi, B.; Gelfi, C.; Righetti, P. G. J. Biochem. Biophys. Methods 1987, 15, 147–161. (4) Wenger, P.; Dezuanni, M.; Javet, P.; Gelfi, C.; Righetti, P. G. J. Biochem. Biophys. Methods 1987, 14, 29–43. (5) Zuo, X.; Speicher, D. W. Anal. Biochem. 2000, 284, 266–278. (6) Ros, A.; Faupel, M.; Mees, H.; van Oostrum, J.; Ferrigno, R.; Reymond, F.; Michel, P.; Rossier, J. S.; Girault, H. H. Proteomics 2002, 2, 151–156. (7) Tran, J. C.; Doucette, A. A. J. Proteome Res. 2008, 7, 1761–1766. (8) An, Y.; Fu, Z.; Gutierrez, P.; Fenselau, C. J. Proteome Res. 2005, 4, 2126–2132. (9) Lim, P.; North, R.; Vigh, G. Electrophoresis 2007, 28, 1851–1859. (10) Cologna, S. M.; Russell, W. K.; Lim, P. J.; Vigh, G.; Russell, D. H. J. Am. Soc. Mass Spectrom. 2010, 21, 1612–1619. (11) Hubner, N. C.; Ren, S.; Mann, M. Proteomics 2008, 8, 4862–4872. (12) Hjerten, S.; Valtcheva, L.; Elenbring, K.; Liao, J. L. Electrophoresis 1995, 16, 584–594. (13) Bossi, A.; Olivieri, E.; Castelletti, L.; Gelfi, C.; Hamdan, M.; Righetti, P. G. J. Chromatogr., A 1999, 853, 71–82. (14) Hjerten, S.; Liao, J.-L. Electrophoresis in low conductivity buffers. U.S. Patent 5,464,517, November 7, 1995. (15) Gelfi, C.; Perego, M.; Righetti, P. G.; Cainarca, S.; Firpo, S.; Ferrari, M.; Cremonesi, L. Clin. Chem. 1998, 44, 906–913. (16) Gelfi, C.; Mauri, D.; Perduca, M.; Stellwagen, N. C.; Righetti, P. G. Electrophoresis 1998, 19, 1704–1710. (17) Fullarto, J. R.; Kenny, A. J. Biochem. J. 1970, 116, 147–149. (18) Shave, E.; Vigh, G. Electrophoresis 2004, 25, 381–387. 8113

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