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Comparison of Fluorescence, Laser-Induced Fluorescence, and Ultraviolet Absorbance Detection for Measuring HPLC Fractionated Protein/Peptide Mixtures King C. Chan, Timothy D. Veenstra, and Haleem J. Issaq* Laboratory of Proteomics and Analytical Technologies, Advanced Technology Program, SAIC-Frederick, Inc., National Cancer Institute at Frederick, P.O. Box B, Frederick, Maryland 21702, United States ABSTRACT: Proteomics is the study of all proteins in a biological sample. High-pressure liquid chromatography coupled online with mass spectrometry (HPLC/MS) is currently the method of choice for proteomic analysis. Proteins are extracted, separated at the protein or peptide level (after enzymatic digestion), and fractions are analyzed by HPLC/ MS. Detection during off-line fractionation is generally conducted using UV-vis, which is not sensitive enough to distinguish fractions having the largest concentration of proteins/ peptides and should not be combined prior to HPLC/MS. To overcome this deficiency, we utilize fluorescence or UV-laser induced fluorescence (UV-LIF) detection for measuring proteins/ peptides during the off-line fractionation. Fluorescence detection allows low-abundance proteins/peptides that contain aromatic amino acids to be measured. In this study, peptide/protein samples fractionated using ion-exchange chromatography were detected using UV absorbance, fluorescence, and UV-LIF. The results indicated that fluorescence and UV-LIF were able to detect the lower abundance proteins/peptides to give a more representative chromatogram, allowing the analyst to decide which fractions should be combined prior to HPLC/tandem mass spectrometry (MS/MS) analysis.
The proteome describes the entire compliment of proteins expressed by a cell at a point in time. Alterations in protein abundance, function, and structure can serve to indicate pathological abnormalities, making the proteome presumably rich in diagnostic and prognostic biomarkers. Electrophoresis and chromatography followed by mass spectrometry (MS) for protein detection and identification are the methods of choice for protein biomarker discovery. Discovering biomarkers in biological specimen in the past few years has focused on developing analytical methods for identifying the maximum number of proteins in cells, serum, plasma, urine, cerebrospinal fluid (CSF), and others. Since blood circulates throughout the body and has intimate contact with almost every cell, it has been the single most studied sample for disease biomarker discovery. The blood proteome contains thousands of proteins of different chemical and physical properties and widely different concentration levels. Most published proteomic studies employ the bottom-up approach developed by Yates and co-workers1 where proteins are extracted, enzymatically digested, and fractionated online by strong cation exchange chromatography (SCX). The individual SCX fractions are then analyzed using reversed phase highpressure liquid chromatography/mass spectrometry (HPLC/ MS). In another approach, initial fractionation is carried out at the protein level without prior enzymatic digestion. Proteins in each fraction are then enzymatically digested and analyzed by HPLC/MS.2 Most published studies employed an off-line SCX This article not subject to U.S. Copyright. Published 2011 by the American Chemical Society
fractionation approach since it is simpler, easier to perform, allows the collection of the largest number of fractions, and does not require any type of sophisticated column preparation.3 Fractions are collected based on a predetermined time period (minutes) with or without detection. The majority of published studies used UV absorption detection, which lacks the required sensitivity to detect low abundance peptides/proteins. Unfortunately, a UV chromatogram does not represent all the proteins/ peptides in a proteome. Fluorescence and ultraviolet-laser-induced fluorescence (UV-LIF) are g100-fold more sensitive than UV, enabling the analyst to (a) detect a greater number of peptides/proteins and (b) combine fractions based on sound empirical data. On the basis of the recorded chromatogram of the SCX fractionation procedure, fractions are analyzed by RP-HPLC/ MS either individually or the weaker fractions are combined to save analysis time and effort. Fractionation prior to separation and MS detection has been used to (a) simplify the complexity of the proteome, (b) allow full utilization of the dynamic range of the system, (c) reduce mass spectral complexity, and (d) allow the identification of the largest number of peptides/proteins. Received: December 13, 2010 Accepted: February 1, 2011 Published: February 14, 2011 2394
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’ EXPERIMENTAL SECTION Materials. All analytical grade chemicals and solvents were purchased from commercial suppliers. Instrumentation and Procedure. HPLC was performed using an Agilent 1100 system. SCX of peptides was achieved with a 1 mm 150 mm column packed with 5 μm particle size polysulfethyl A material (PolyLC, Columbia, MD) using the following step gradient: 3% B at 0 min, 10% B at 40 min, 60% B at 86 min, and 100% B at 87 min. Solvent A was 25% acetonitrile and solvent B was 25% acetonitrile containing 0.5 M ammonium formate buffer, pH 3. The flow rate was 50 μL/min. Protein fractionation was carried out using a 4.6 mm 200 mm weak anion-exchange column from PolyLC with the following step gradient: 0% B for 10 min, then 80% B at 70 min, 100% B at 80 min for 16 min. Solvent A was 5% acetonitrile, and solvent B was 5% acetonitrile containing 0.6 M ammonium acetate, pH 7.2. The flow rate was 1 mL/min. Online UV absorption and fluorescence/LIF detections were carried out serially on the same sample. The LIF detector was constructed in-house using a solid-state UV laser (266 nm) purchased from Uniphase (San Jose, CA). Fluorescence and UV detectors were purchased from Agilent Technologies (Palo Alto, CA). Fluorescence was monitored at λexcitation 280 nm and λemission 340 nm, while UV absorption was monitored at 280 nm. Extracted proteins were tryptically digested in buffered methanol and fractionated by SCX according to a published procedure.4 Fractions were collected every minute with the aid of an automatic microfraction collector (Foxy Jr., Isco, Lincoln, NE), which collects samples by time, number of drops, or an external signal.
’ RESULTS AND DISCUSSION The analysis of a proteome involves the separation and quantitation of as many proteins as possible in a biological sample such as cell, blood, tissue, etc. The analysis is carried out, in most reported studies, by first enzymatically digesting the extracted proteins followed by two-dimensional liquid chromatography in which the first dimension is generally off-line or online ion exchange (IEX) chromatography, reversed phase (RP) chromatography, or another mode of separation. What is common to any of these separation methods is the use of UV detection. The third step in this process is separation of the collected fractions, detection, identification, and quantitation using LC/tandem mass spectrometry (MS/MS). In the off-line approach, fractions are collected every minute or few minutes resulting in tens of samples, depending on the gradient time and the study design. To save analysis time, fractions containing low amounts of protein, based on the UV/LC chromatogram, are combined. The UV detection is carried out at 214 or 280 nm. Detection at low UV wavelengths introduces errors since many compounds absorb at 214 nm; however, absorption measurements using 280 nm results in weaker signals. Combining fractions based on the UV/LC chromatogram or without detection is not ideal and may result in combining many fractions that actually contain a high concentration of peptides but only provide a weak UV signal. Analyzing such combined fractions using LC/MS/MS in a data-dependent mode will limit the percentage of unique peptides that can be selected for identification. A better approach for off-line fractionation is to replace the
Figure 1. High-performance liquid chromatograms of intact proteins extracted from a cell lysate monitored using fluorescence (upper trace) and UV absorbance (lower trace) detection. Experimental conditions are as described in the Experimental Section.
Figure 2. High-performance liquid chromatograms of tryptic peptides prepared from a proteome sample extracted from a cell lysate monitored using LIF (upper trace) and UV absorbance (lower trace) detection. Experimental conditions are as described in the Experimental Section.
UV detector with a more sensitive detection system such as fluorescence or UV-laser-induced fluorescence. In our laboratory, we built a LIF detector using a KrF pulsedUV laser and applied it for the detection of native peptides and proteins fractionated using capillary electrophoresis.5 In the present study, the KrF laser was replaced with a solid state UV laser6 and applied for the detection of peptides separated by SCX. The weak anion-exchange chromatography (WAX) chromatograms of a cell lysate fractionated using UV detection at 280 nm and fluorescence detection at λexcitation of 280 nm and λemission at 340 nm are shown in Figure 1. Identical experimental conditions were used to generate both chromatograms. Since ammonium formate, which absorbs light with a wavelength of 214 nm, was added to mobile phase B, 280 nm was used in both the UV and fluorescence studies. While the overall differences are clear, discrepancies within particular regions of the chromatograms are striking. For example, while the UV chromatogram suggests very little protein is eluting between 45 and 90 min, the fluorescence trace shows that in fact a significant amount of protein is present during this period. If only UV detection were 2395
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utilized, it may be tempting to pool all of the fractions collected after 45 min into a small (e.g., 2-3) number of aliquots for downstream LC/MS/MS analysis. Normally the chromatogram of a fractionated digested proteome (i.e., peptides) is examined prior to deciding to study each fraction or combined specific fractions for downstream LC/MS/ MS analysis. The chromatograms of a tryptically digested proteome monitored using UV absorption and LIF detections are shown in Figure 2. The UV chromatogram provides essentially no useful information that enables intelligent pooling of the fractions. In particular, the region after 46 min is useless owing to the rising absorption measurement produced from increasing interference resulting from mobile phase B. While the information within the UV chromatogram is limited, the LIF chromatogram enables intelligent pooling of fractions prior to LC/MS/ MS analysis. For example, an investigator may decide to pool fewer samples collected between 15 and 30 and 50-60 min (where signal intensity is high) than between 30 and 50 min (where signal intensity is low).
’ CONCLUSIONS In this study, fluorescence and LIF detection were compared to UV absorption detection for monitoring the fractionation of complex protein and peptide mixtures using ion exchange chromatography. Fluorescence detection provides much greater information enabling fractions to be combined prior to LC/MS/ MS analysis based on sound empirical data. This information can increase the efficiency of the overall study and prevent the pooling of samples with inordinate protein content being analyzed using LC/MS/MS. ’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Fax: þ301 846 6037.
’ ACKNOWLEDGMENT This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the United States Government. ’ REFERENCES (1) Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R.,, III Nat. Biotechnol. 1999, 17, 676–682. (2) Hood, B. L.; Zhou, M.; Chan, K. C.; Lucas, D. A.; Kim, G. J.; Issaq, H. J.; Veenstra, T. D.; Conrads, T. P. J. Proteome Res. 2005, 4 (5), 1561–1568. (3) Issaq, H. J.; Conrads, T. P.; Janini, G. M.; Veenstra, T. D. Electrophoresis 2002, 23 (17), 3048–3061. (4) Blonder, J.; Chan, K. C.; Issaq, H. J. Nat. Protoc. 2006, 1, 2783–2790. (5) Chan, K. C.; Janini, G. M.; Muschik, G. M.; Issaq, H. J. J. Liq. Chromatogr. 1993, 16, 1877–1890. (6) Chan, K. C.; Muschik, G. M.; Issaq, H. J. Electrophoresis 2000, 21, 2062–2066. 2396
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