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Sample treatment for protein identification by mass spectrometry-based techniques
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Trends in Analytical Chemistry, Vol. 25, No. 10, 2006
Sample treatment for protein identification by mass spectrometry-based techniques D. Lo´pez-Ferrer, B. Can˜as, J. Va´zquez, C. Lodeiro, R. Rial-Otero, I. Moura, J.L. Capelo Rapid identification of proteins is of primary importance for the analytical community. Protein-biomarker discovery for medical diagnostics or pharmacological purposes is becoming one of the hottest research topics. Moreover, rapid identification of proteins can help unambiguous bacterial and virus detection. In addition, the fast identification of bacteria can be used to beat bioterrorism. As a consequence, new analytical methodologies have emerged recently with the aim of making protein analysis as fast and as confident as possible. In this article, we critically review the new trends in sample treatment for protein identification and comment on the prospects for the future in this promising analytical area. ª 2006 Elsevier Ltd. All rights reserved. Keywords: Biomarker; Protein; Rapid identification; Sample treatment Abbreviations: ALS, Acid-labile surfactant; BSA, Bovine serum albumin; BTRFE, Bovine apotransferrin; CE, Capillary electrophoresis; CID, Collisioninduced dissociation; DTT, Dithiothreitol; IAA, Iodoacetamide; 2DE, Two-dimensional electrophoresis; ESI, Electrospray ionization; HIFU, Highintensity focused ultrasound; hTf, Human transferrin; IEF, Isoelectric focusing; LC, Liquid chromatography; MALDI-TOF, Matrix-assisted laser desorption/ionization – time-of-flight; MAPED, Microwave-assisted protein enzymatic digestion; MS, Mass spectrometry; PAGE, Polyacrilamide gel electrophoresis; PFF, Peptide-fragment fingerprinting; PMF, Peptide-mass fingerprinting; Q-TOF, Quadrupole time-of-flight; RP, Reversed phase; SCX, Strong cation exchange; SDS, Sodium dodecyl sulphate; TCA, Trichloroacetic acid.
1. Introduction D. Lo´pez-Ferrer, J. Va´zquez Centro de Biologı´a Molecular ‘‘Severo Ochoa’’-CSIC, 28049, Cantoblanco, Madrid, Spain B. Can˜as Departamento de Quı´mica Analı´tica, Facultad de Quı´micas, Universidad Complutense de Madrid, Madrid 28040, Spain C. Lodeiro, R. Rial-Otero, I. Moura, J.L. Capelo* REQUIMTE, Departamento de Quı´mica, Faculdade de Ciencia e Tecnologia, Universidade Nova de Lisboa, Monte de Caparica, 2829-516, Caparica, Portugal
*
Corresponding author. Tel.: +351 212 949 649; Fax: +351 21 294 8550; E-mail: [email protected]
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The identification of proteins is becoming more important in the scientific community due to emerging issues critically significant to society. Many hereditary diseases, some types of cancer and common diseases, such as diabetes, can be differentiated on the basis of the expression of certain proteins, known as protein biomarkers. As a result, disease screening and medical diagnosis take advantage of mass spectrometry (MS)-based technological improvements that allow ultra-fast protein identification [1,2]. Fast identification of protein biomarkers to identify bacteria is helping to reduce morbidity and mortality significantly across the globe [3,4]. Furthermore, rapid bacterial assessment through protein identification will help civil and military defense organizations to
0165-9936/$ - see front matter ª 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2006.05.015
Trends in Analytical Chemistry, Vol. 25, No. 10, 2006
beat bioterrorism [5], and recent literature suggests that protein identification can also be applied to assessing viruses [6]. The main limiting steps for rapid protein identification are (i) the number of protein entries in database; and, (ii) sample treatment. The second limitation is due to the handling protocols commonly used for protein identification. Traditionally, these protocols are tedious, with many single steps. In addition, from four hours to overnight is the time usually required for enzymatic digestion for protein identification. Most improvements reported in literature in the past five years have been devoted to enhancing the throughput of sample treatments. The main aims of this article are: (i) to review advances in sample treatment for fast protein identification; (ii) to discuss the parameters affecting each new methodology along with their advantages and drawbacks; (iii) to show current applications of protein identification; and, (iv) to comment future prospects.
2. Enzymatic digestion of proteins Protein isolation or purification is the first step in protein identification. The second step is specific cleavage of the peptide bonds in a protein to produce peptides, which is mainly done using enzymes, obtaining an enzymespecific pool of peptides; this procedure is known as protein digestion. The characteristic pool of peptides produced is then analyzed by MS-based techniques, such as matrix-assisted laser desorption/ionization time-offlight MS (MALDI-TOF-MS) or liquid chromatographyMS (LC-MS), to obtain accurate measurements of their masses. In most cases, this set of masses is enough for unambiguous identification of the protein. This methodology is known as peptide-mass fingerprint, PMF [7], which involves comparing the experimental masses from the peptides produced by the digested protein, and those produced by in silico, theoretical, digestion of all the proteins in a particular database. Special search programs [8], known as search engines, are used for this purpose. Here it is found the Achilles Heel for fast protein identification: the sequence of the protein to be identified must be included in the database; otherwise, PMF fails to identify the protein. 2.1. Types of enzymes: intended effects As stated above, peptides are produced from proteins through enzymatic digestion. Nevertheless, this is not the only way to digest proteins, since special chemical reagents, such as cyanogen bromide [9], can be also used. The choice between chemical or enzymatic protein
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cleavage will depend on several factors (e.g., the type of protein, its sequence and the purpose of the analysis). Enzymatic digestion presents several advantages for PMF, mainly due to the size of the peptides produced, which is very compatible with their analysis by MS. The enzymes commonly used for protein digestion comprise a group of proteins called proteases, which are hydrolytic enzymes that act on the amide bonds at specific positions in the protein under digestion. There are different types of proteases; the most frequently cited in the literature belong to one of the following groups [10]: (i) serine; (ii) aspartic; (iii) cysteine; or, (iv) metalloproteases. Differences among proteases account for (i) the mechanism of protein cleavage, and (ii) conditions for optimum performance. Concerning mechanisms of protein cleavage, when the aim of the analysis is protein identification, trypsin, a serine protease, is the most widely used enzyme to perform protein digestion, since it presents the following advantages: (i) it cleaves exclusively after arginine or lysine residues, which are present in proteins at the approximate rate of one residue per every 10–12 amino-acids. Peptides produced have an average size of 800–2000 Da, very adequate for MS analysis; and, (ii) peptides resulting from trypsin digestion produce very homogeneous fragmentation under collision-induced dissociation (CID), a special methodology in MS, so fragmentation spectra of only one tryptic peptide may give enough information to identify a protein, if it is included in the database. As for conditions for performance, for most enzymes, activity is temperature dependent until a maximum is reached, when the enzyme is no longer stable. Furthermore, enzymes, as many other proteins, are stable only over a limited range of pH; outside this range, changes in the charges of ionizable amino-acid residues may result in modification of the tertiary structure of the protein and eventually lead to enzyme denaturation and inactivation. There is a pH value at which the activity of the enzyme reaches a maximum or a plateau, so protein digestion is usually performed in a buffered medium. Enzyme concentration also affects the process, so it needs to be optimized. Three different strategies can be used for protein digestion using enzymes, as summarized in Fig. 1. In the first procedure, protocol A, proteins initially isolated by SDS-PAGE or two-dimensional electrophoresis (2DE), are digested in situ, inside the gel structure. This methodology is known as in-gel protein digestion [11]. In the second approach, protocol B, cell extracts, comprising protein mixtures with different degrees of complexity, are enzymatically digested, sometimes without previous purification. This methodology is called in-solution digestion [12]. This procedure has several advantages (e.g., compatibility of protein identifications carried out at the same time). In the third strategy, protocol C, the http://www.elsevier.com/locate/trac
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solution containing a protein mixture is passed through a column filled with a chromatographic packing containing an immobilized enzyme. This is the in-column digestion methodology and is very useful for simple mixtures of proteins in solution [13]. Using the in-column approach, it is possible to couple protein digestion, peptide separation and MS analysis, so facilitating the complete analysis on-line. Table 1 shows the advantages and drawbacks of these three sample treatments, which we treat in detail below.
Cell lysis
Protein Cell Extract
Protocol A
Protocol B
Protocol C
Protein denaturation
Protein precipitation
HPLC protein separation
1D or 2D-SDS-PAGE protein separation
Protein denaturation
Isolated Proteins
Isolated Proteins
Reduction & alkylation Steps
Enzyme digestion (4-12 hours, 37 ºC)
Pass through immobilized trypsin cartridges
Spot excision
Enzyme digestion (4-12 hours, 37 ºC)
Stop hydrolysis (acid addition) Peptide Pool
Stop hydrolysis (acid addition)
Complex peptide pool
Peptide Pool 2D-HPLC peptide separation
HPLC peptide separation
Mass Spectrometry Analysis
Protein Identification
Figure 1. Enzymatic protein-digestion protocols. Protocol A corresponds to the classical protein analysis, in which proteins are initially separated according to their isoelectric point and then according to their MW. Protocol B corresponds to the typical gel-free approach, in which proteins are digested prior to any separation step and then the whole peptide pool is separated using multi-dimensional chromatography in tandem with mass spectrometry. Protocol C represents the new trends in protein digestion using immobilized trypsin. Firstly, proteins can be separated using different chromatography steps, and are then digested in-column, and, finally, the peptide pool is separated and analyzed.
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2.2. In-gel protein digestion This is by far the most tedious and time-consuming approach, and most dedicated advances in recent literature are devoted to speeding up this methodology, as we will show later. Fig. 2 shows a general scheme for this sample treatment, in which the common steps for in-gel protein digestion are presented in conjunction with their intended effects. As can be seen, there are many steps that must be followed to digest a protein correctly. First, proteins must be separated and purified. The most frequent separation strategy used for complex protein mixtures is one-dimensional electrophoresis or 2DE, as previously reviewed [14]. Depending on the sample, it may be necessary to concentrate and eliminate interfering substances before electrophoretic separation. In such a case, it is good practice to precipitate protein extracts with a mixture of trichloroacetic acid (TCA)/ acetone [15]. To ensure efficient, reproducible separation in the gel, protein extracts must previously be denatured in urea/thiourea at high concentrations, 8–9 M. In addition, thiourea increases the solubility of hydrophobic proteins [16]. To run the first dimension of 2DE, samples are applied to isoelectric focusing (IEF) gel strips containing ampholites, substances with different pKa values that create a pH gradient along the gel. Nowadays, gel strips for the first dimension are prepared with immobilized ampholites, in the form of acrylamide derivatives, called immobilines [17], which dramatically increase reproducibility. Once the first dimension is completed, and to prevent further re-oxidation, protein-cystine residues must be reduced using dithiothreitol (DTT) and the resulting cysteines blocked with iodoacetamide (IAA). After focusing, IEF strips are soaked in SDS to prepare proteins for separation by molecular masses (MW) in the second dimension. When the separation is complete, proteins are visualized in the gel by staining with Coomassie Blue, fluorescent dyes, or with MS-compatible silver nitrate [18]. The result of the 2DE separation procedure is shown in protocol A (Fig. 1), in which each dark spot corresponds, theoretically, to a protein.
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Table 1. Analytical parameters for enzymatic protein digestion Variable
In-gel digestion
In-solution digestion
In-column digestion
Treatment time Handling Sample throughput On-line application Protein-digestion yield Robustness Enzyme-to-protein ratio allowed Cost
up to 12 h high low no low low low low
up to 12 h medium medium yes medium high very low low
up to 10 min. low high yes high medium medium medium
Further treatment may require intact proteins to be eluted from the gel. Proteins may be extracted from the gel by different techniques (e.g., (i) passive elution [19], (ii) electroelution [20], or (iii) the ultrasonic-assisted elution [21]). Once eluted, proteins are digested in solution and then analyzed by PMF. Nevertheless, protein elution from gels is troublesome, being protein dependent and, generally, with very low recovery yields. This is why most researchers in the field of protein identification prefer to digest proteins in situ, inside the gel, without previous elution. This procedure is based on the peptides are most easily eluted from gels than the original, undigested proteins, so the problems associated with protein elution from gels are overcome. As a result, robust protocols have been developed for in-gel digestion [22]. In addition, automated protein digestion is available in many proteomic facilities. To proceed with in-gel digestion of proteins, separate protein bands or spots are cut from the gel and de-stained, following procedures that depend on the staining procedure. There are protocols for removing Coomassie-blue [23] or silver [24] from pieces of gel. Once de-stained, and to ensure adequate digestion of the protein inside the gel, the following parameters must be optimized: (i) accessibility of the enzyme to the cleavable bonds may sometimes be low, because proteins are trapped inside the gel structure, so it is very important to maintain an adequate enzyme/substrate ratio to maximize in-gel digestion, this ratio being much higher than that needed for in-solution digestion; (ii) solvent composition inside the gel must be strictly controlled, in order to achieve a good digestion yield; for this purpose, gel slides must first be dehydrated using acetonitrile and dried in a speedvacuum, the objective of this treatment being to facilitate in-gel penetration of the solution containing the enzyme, which is added after gel drying; and, (iii) the pH and temperature of the solution must be controlled; otherwise, enzyme efficiency can be lowered.
From a few hours to overnight incubation at 30C or 37C is usually enough for complete protein digestion. Finally, the supernatant containing the peptides is acidified to stop the digestion process and subjected to MS analysis, generally using a MALDI-TOF instrument [25] for protein identification by PMF. The relation between the amino-acidic sequence found experimentally and the theoretical sequence of the assigned protein, expressed as percentage, is known as the sequence coverage of the PMF, and is crucial for trust in protein identification. The in-gel digestion protocol presents several drawbacks, namely (i) trapping protein substrates in the gel makes some peptide bonds inaccessible to the enzyme, and (ii) not all the peptides produced during digestion can diffuse freely from the gel. This explains the difference in results between in-gel and in-solution digestion. Table 2 shows selected examples of in-gel protein digestion. 2.3. In-solution protein digestion Shotgun proteomics, initially developed by Link et al. [12], has become very popular in the past few years. It is based in whole-extract protein digestion in solution. This procedure was extensively used in the analysis of isolated proteins, but is now applied to complex protein mixtures. Protein composition and their particular chemical and physical properties are very diverse. As a result, the degree of protein digestion and the type and quantity of peptides obtained differ greatly from one protein to another. When working with this procedure, it is important to keep in mind the following: (i) more abundant proteins are usually easier to identify; (ii) to facilitate identification of minor proteins, some fractionation (e.g., organelle purification) is needed; and, (iii) variation in the yield of digestion from sample to sample can occur because of (1) heterogeneity in the matrix, (2) the quantity and number of proteins, and (3) physical and chemical properties of the proteins.
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Classical Protein Analysis Treatment for 1D Gels: total time 20-24 h.
Purification
5µl of protein + 5µl of Sample buffer for SDS-PAGE prepared as follows: tris-base 0,5M at pH=6,6–6,8 (5ml) +10% SDS (8ml) + β -mercaptoethanol (1ml) + glycerol (2ml) + bromophenol blue (4mg) + H2O till 20ml of total volume
Boiling in water bath for 5 min
Reduction of disulfide bonds (βmercaptoethanol) Visualize the sample electrophoretic front (Bromophenol Blue) Optimize pH for trypsin activity (TrisBase 0,5M) Remove protein charges (SDS)
Protein denaturation
Electrophoresis (60/70 min) at constant voltage of 120V and 400mA
Protein separation and purification
Staining with coomasie blue R-250 (1g) + glacial acetic acid (15ml) + methanol (90ml) + H2O till 200ml of total volume
Visualize the proteins bands through staining
De-staining with a decolouring solution prepared with: glacial acetic acid (75ml) + methanol (450ml) + H2O till 1000 ml of total volume
Remove the excess of colouring solution
Cut off protein spots and slight slicing the gel with the scalpel
Wash with water (3 x 100µl) and acetonitrile (3 x 100µl)
Treated with DTT (40 µl, 25 min) and IAA (30µl, 35 min)
Sample Preparation
Intended Effect
Wash with water (3 x 100µl) and acetonitrile (3 x 100µl)
Dry in a dried speed- vacuum
Add trypsin (15 µl) and left in a ice bath for 1 h
Facilitate trypsin penetration into the gel
Removal interferences
Reduction and alkylation di-sulphide bonds
Removal interferences
Dehydrate the gel
Penetration of the enzyme into the gel without digestion
Add NH4HCO3 buffer 12,5mM (10µl)
Maintain pH during incubation time
Incubate at 37ºC for 4 h or overnight. (HIFU sonication for 2 minutes)
Digestion of proteins into peptides
Add formic acid 0,3% (15µl)
Stop trypsin activity
Figure 2. Detailed description of an in-gel protein digestion protocol.
As a general rule, to overcome these drawbacks, protein extracts are: (a) first precipitated using cold acetone or TCA; (b) after precipitation, and in order to make the proteins more accessible to the action of the enzymes, proteins are re-suspended in ammonium bicarbonate buffer supplemented with 8 M urea or another chaotropic agent (e.g., guanidine hydrochloride) to break intra-molecular forces and denature proteins; and, finally, 1000
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(c) to prevent protein renaturation before enzymatic attack, reduction and alkylation of protein disulfide bridges are commonly done using DTT and IAA, although other reagents are also available for the same purposes. As mentioned above, trypsin is the enzyme chosen for protein digestion because of its specificity [22]. However, some in-solution protocols [26,27] make use of two digestion steps, first Lysine-C (Lys-C) and then trypsin, because Lys-C fully preserves activity in the presence of
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Table 2. Selected sample treatments for enzymatic protein digestion Analytical task In-gel protein digestion Identification of proteins from 2D-PAGE with a new acid-labile surfactant
Comparison of protein-precipitation methods for sample preparation prior to proteomic analysis
Protein in-gel digestion for proteome characterization
Comparison of different staining procedures for MS
In-solution protein digestion A largely unbiased method for rapid, large-scale proteome analysis
Proteomic mapping of brain plasma-membrane proteins
A simple, rapid method for characterizing hydrophobic integral membrane proteins.
Fast proteolytic digestion coupled with organelle enrichment for proteomic analysis of rat liver.
In-column protein digestion Ion-electrospray capillary proteolysis
Analytical techniques/comments
Ref.
MALDI-TOF-MS / Results indicate that substituting SDS by ALS, the numbers of peptides detected by MALDI-TOF-MS are significantly increased, especially for proteins of low abundance Spot identity of gel-slides was done by similarity with previously scanned gels / Four protein-precipitation methods were compared: (i) trichloroacetic acid, (ii) acetone, (iii) chloroform/methanol, and (iv) ammonium sulfate. Precipitation of proteins with TCA and acetone and ultra-filtration result in an efficient sample concentration and desalting for proteomic analysis MALDI-TOF-MS / A study was conducted to obtain a fast, reliable approach to enhance in-gel protein digestion and high peptide recovery. MOWSE score obtained was twice that of the standard protocol MALDI-TOF-MS and nano-LC-MS2(Q-TOF) / The following staining procedures were compared: (i) colloidal Coomassie blue, (ii) Daiichi Silver, (iii) SYPRO Orange, (iv) SYPRO Red, (v) SYPRO Tangerine, and (vi) SYPRO Ruby. Best results with SYPRO Red and Orange
[30]
Multidimensional LC-MS2 / A total of 1484 proteins were detected and identified (proteome of Saccharomyces cerevisiae strain BJ5460). 131 proteins with three or more predicted transmembrane domains. Complex peptide mixtures were loaded separately onto a biphasic microcapillary column packed with strong cation exchange (SCX) and reverse-phase (RP) materials LC-MS2 (Q-TOF) and LC-MS2 (Ion-Trap-FT) / A novel method for extraction and fractionation of membranes, onmembrane extraction followed by on-membrane digestion and diagonal separation of peptides. 862 proteins from 150 mg of mouse brain cortex and 1685 proteins from 15 mg of hippocampus were identified Micro-capillary LC-MS2 (lLC-MS2)/A detergent and cyanogen bromide-free method for integral membrane proteomics was applied to Halobacterium purple membranes and the human epidermal membrane proteome. From human epidermal sheets, a total of 117 proteins were identified LC-MS2(Ion-Trap) / The use of an acid-labile surfactant (ALS) as an alternative to urea denaturation was demonstrated by comparison of the same sample treated with (i) both organelle enrichment and whole-cell lysate or (ii) reduction, alkylation, and urea denaturation. Number of peptides obtained: 694 for urea, 674 for ALS; number of proteins 225 for urea, 229 for ALS
Q-TOF-MS (Positive ion mode (ESI (+)). / The ionization source was set at 80C. 10 pmol/lL of BSA or BTRFE dissolved in (5–10 mM NH4HCO3 + 10–20% methanol) plus final Trypsin or endoproteinase Glu-C concentration of 0.02–0.4 pmol/lL, respectively. The mixture was transferred to the electrospray capillary without further treatment. Sequence coverage: >40% with trypsin and 100% with Glu-C. Validation with elephant milk protein preparation: five proteins were correctly identified
[15]
[23]
[18]
[27]
[26]
[29]
[48]
[49]
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Table 2 (continued) Analytical task
Analytical techniques/comments
Ref.
Chip-based nanovials proteolytic digestion
CE; MALDI-TOF / Two different protocols are described for enzyme (trypsin, chymotrypsin) immobilization in nanovials. Nanoliter-scale enzymatic digestion. Digestion time 3–4 h at 37C. Chip re-utilization impossible MALDI-TOF-MS / Three water mixtures (methanol, isopropanol, acetonitrile) were used for digestion of human transferrin (hTf). Digestions with methanol produced fewer missed cleavages, whilst, with acetonitrile, they produced most peptide and intense mass spectra. Digestion of low nanomolar myoglobin was OK LC-ESI-MS2 / Steps: (i) capture the proteins on the column packing material; (ii) fill column with enzyme for protein digestion; (iii) wash column: elution of enzyme excess; (iv) elute peptide fragments. Total time: 80 min., each cycle. 3 HPLC pumps, 2 C8 columns and 2 micro-digestion columns
[50]
On-column digestion in aqueous-organic solvents
Digestion with immobilized proteins
8 M urea, the accessibility of this enzyme to peptide bonds being greater than trypsin; this activity is possible only after a urea dilution to 2 M. Protein digestion is usually performed for 12–24 hours, due to protein heterogeneity in samples. Finally, the reaction is stopped by adding an acid, such as trifluoroacetic or formic, to a pH of 2–3. Then, the digest can be directly analyzed by 2DE-LC-MS2. In some cases, lyophilization or evaporation in a speed vacuum can be used for (i) further sample concentration or (ii) sample storage until analysis; in this case, temperature preservation is needed at, or even below, 20C. Hydrophobic membrane proteins, with low solubility in water, represent a special case. These proteins play important roles in various cellular processes, such as signal transduction and cell adhesion. Cyanogen bromide at high concentrations of acid [28] or tryptic digestion at high concentrations of methanol [29] have been reported in the literature to handle membrane proteins successfully. A promising methodology for membrane proteins uses acid-labile surfactants (ALSs) [30]. Furthermore, using ALS steps (e.g., urea denaturation of proteins), disulfide reduction/alkylation may be eliminated. In addition, proteolytic digests performed in the presence of ALS are easy to recover and do not need desalting steps for further chromatography and MS analysis. Table 2 shows selected examples of in-solution protein digestion. The complex mixture of peptides produced by the insolution digestion procedure is separated using multi-dimensional LC, coupled to electrospary ionization (ESI)-MS2 [12]. Peptides are identified by their fragmentation spectra, in a similar way to PMF, but using specialized software that compares the experimental fragmentation pattern of a peptide with that generated in silico from all the proteins in a database. This procedure is known as peptide-fragment fingerprinting (PFF) [31]. 1002
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[36]
[34]
2.4. In-column protein digestion Usually, tryptic protein digestion is done either in gel or in solution, as described above, although these methodologies have several drawbacks [32]: (i) long digestion time (typically >5 h); (ii) low trypsin-to-substrate ratios are allowed to avoid the production of interfering peptides from trypsin autolysis; (iii) low digestion yields obtain when the substrate concentration is in the low-micromolar range; (iv) time-consuming handling; and, (v) peptide losses by adsorption can occur. To overcome these problems, proteolytic digestion may be performed in solid supports containing immobilized enzymes. This procedure, called in-column digestion, has the following advantages: (i) lower digestion times; (ii) increased digestion ratios; and, (iii) incorporation into multi-dimensional separation procedures for automated proteomics. Several companies provide cartridges with highdensity immobilized trypsin (e.g., the Poroszyme cartridge that can be used into integrated systems including MS detection for peptide identification [33,34]). However, high cost, limited catalytic activity and autolysis are still limitations that need to be overcome. In this methodology: (i) multi-dimensional chromatography is used to purify proteins from mixtures; (ii) the separated proteins are then digested by passing them through immobilized trypsin cartridges; and, (iii) the peptides formed are separated by reverse phase HPLC. As a result, integrated protein identification is possible. Trypsin can be immobilized chemically, trapped or physically adsorbed. The packing material used for enzyme immobilization is usually made of silica or
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polymeric material, and is packed as particulate or as monolithic columns. Monolithic columns are preferred because of their low cost and relatively little resistance to solvent flow, and that allows fast analysis and higher throughput. These properties are due to their open, rigid structure. The properties of the material used for enzyme immobilization, such as shape or porosity, affect the performance of the digestion reactor. The review based on chemical reactors for on-line systems by Massolini and Calleri [35] is highly recommended. Freije et al. [32] reported enhancement in proteolysis activity when trypsin reactions involved chemically modified (acetylated) trypsin. In addition, they found fewer interfering trypsin autolysis products. The use of water mixed with organic solvents may help in incolumn digestion of resistant proteins, as was demonstrated by Slysz and Schriemer [36]. Furthermore, a complex system (Fig. 3, coupling three HPLC pumps, two digestion columns, two separation columns and three injection valves) has been described in the literature [34]. This analytical approach may be necessary for the following reasons: (i) to prevent losses of hydrophilic peptides after their elution from the trypsin cartridge; (ii) to trap peptides in a capillary column before separation by one-dimensional or two-dimensional chromatography; and, (iii) to avoid detrimental signal suppression in the ESI process by salts by installing a desalting column. Table 2 shows selected examples of in-column digestion.
3. Accelerating protein digestion As stated above, the most recent research reported in the literature has been devoted to the acceleration of the enzymatic digestion process, since it is the most timeconsuming step in identifying proteins. First trials were done taking advantage of basic concepts in enzymology, such as the role of enzyme concentration and the influence of temperature on the rate of the reaction. Using these concepts, a fully enzymatic in-gel protein digestion was achieved in less than 1 hour [37]. There may also be improvements in kinetics, when the digestion is subjected to microwave energy. Recently, an ultra-fast method was developed using high-intensity, focused ultrasound (HIFU). We will comment on both of these methodologies below. 3.1. Microwaves Microwave-assisted protein enzymatic digestion (MAPED) is useful for in-solution [38] or in-gel [39] digestion and may be also applied to complex protein mixtures [40]. Using this methodology, digestion times required to get a complete digestion are up to 20 min. Low irradiation power is used; below 30% of the total nominal power of the microwave oven is recommended; otherwise, temperature control becomes difficult. Critical parameters to be optimized in this procedure are (i) irradiation time, (ii) temperature, and (iii) power. 3.2. Ultrasound The application of HIFU to sample treatment for fast protein identification was first developed by Lo´pez-Ferrer
Digestion Column Colum B AS-HPLC -HPLC 1 system
2 3
1
Separation Column 2
4 10
5 9
8
7
Waste
2
1
6
3 6
AS-HPLC 2 system
Digestion Column A
1 5
Q1
2 3
Ion Prec . C. Cell
4
4
Q2
Q3 LIT
5 6
HPLC pump 3
Waste Mass Spectrometer
Separation Column Colum 1
Figure 3. An on-line protein digestion system. comprising three HPLC pumps (two gradient and one isocratic), three switching valves, and four columns (two used for protein digestion and two other for peptide separation). The whole system is connected on line to a mass spectrometer. To increase throughput of the analysis, each gradient HPLC pump is connected to one digestion column and one separation column through switching valves. These are synchronized to separate (separation-column 1) the digested peptides coming from digestion-column A using one of the gradient HPLC systems. In the meantime, the second gradient system delivers a protein mixture to digestion-column B, while separation-column 2 is being conditioned with the solvent delivered by the isocratic pump. (Adapted from [34], courtesy of The American Chemical Society).
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et al. [41]. Fig. 4 shows HIFU/enzymatic digestion in progress along with the main characteristics. In in-solution protein digestion, the procedure entails sonication with HIFU of small volumes of sample, typically 20–50 lL while the trypsin digestion proceeds. The HIFU treatment probably boosts enzyme-substrate kinetics. The procedure is completed in less than 15 s. In in-gel protein digestion, slides of gel containing protein can be subjected to the same procedure: once excised, the gel piece is introduced in an Eppendorf cup and a small amount of a buffer containing the enzyme is added. The liquid jets produced by the HIFU act as microsyringes [42], delivering the enzyme into the gel and making the protein digestion faster than with any other methodology for in-gel protein digestion: 15–30 s. In addition, the amount of enzyme necessary is three times less than that needed without ultrasonic energy. The mechanical erosion of the gel surface caused by the cavitation associated with ultrasonication enhances peptide release from the gel. This finding deserves more research and is currently being developed in our laboratory. Ultrasonic probes specially developed for small volumes, 10–100 lL, must be used when only minute amounts of protein sample are available. In addition, the use of small sample volumes helps (i) to concentrate the sample, (ii) to reduce time of treatment when the speed vacuum or lyophilization is expected to be used, and (iii) to reduce the amount of enzyme trypsin used. The following parameters must be addressed to optimize performance of this methodology: (a) sonication time; (b) sonication power; (c) sonication amplitude;
1.5 or 0.5 ml reaction cup Ultrasonic probe Cavitation effects
Variables: (i) (ii) (iii) (iv) (v) (vi) ((vii))
Gel slide stained with comassie blue
Probe diameter Sample volume Sonication time Sonication amplitude Enzyme concentration Temperature Sonication power
Figure 4. Enzymatic in-gel protein digestion accelerated by highintensity focused ultrasound (HIFU).
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(d) sample volume; (e) enzyme-to-substrate ratio; (f) probe diameter; and, (g) temperature.
4. Future prospects We expect more research on accelerating chemical reactions using either microwave or ultrasonic energy. As far as microwave energy is concerned, we expect more research to be devoted to the domestic microwave oven, since these devices have not yet not been tried, probably because temperature control is difficult. However, domestic microwave ovens are very cheap, and on-line digestion procedures could be developed so that temperature control can be minimal [43]. We also expect more literature to report on-line procedures in conjunction with microwave energy. Regarding ultrasound, the following research is needed: (i) comparison of sonication probes from different manufacturers in order to assess the robustness of the procedure; (ii) assessment of new ultrasonic devices, such as (a) glass sonication probes [44], (b) sonication baths with different working wavelengths [44], or (c) new powerful sonoreactors [45]; (iii) applicability of the procedure to the recently developed multi-probe sonicators [46], since a minimum of 12 samples can be treated at once and protein digestion can be performed in seconds, thus allowing easy implementation of this procedure in robotic platforms for enzymatic digestion. The possibilities of high sample throughput along with its easy implementation in robotic devices gives this ultrasonic procedure an excellent future in the biotechnology industry and in proteome research. There is one other consideration: the enhancement of enzymatic protein digestion by ultrasonication meets the requirements of analytical minimalism, as proposed by Halls [47]: (1) low cost; (2) low sample requirements; (3) low reagent consumption; and, (4) low waste production.
5. Conclusions Protein identification is an issue of primary importance for the analytical community, its applications covering research areas as diverse as diagnosis of disease and defense against bioterrorism, and demanding the presence of analytical chemists. Improvements in sample treatment achieved using microwave energy or HIFU have changed the methodologies in such a way that protein identification can be
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done in less than 12 h, avoiding previous slow, tedious procedures. We expect protein digestion to be enhanced in the immediate future so that the limiting step in protein identification will be the MS technique chosen for protein analysis rather than the sample treatment. Acknowledgements We thank Antonio Ramos-Ferna´ndez for helpful advice and discussion. This work was supported by grants CICYT BIO2003-01805 and CAM 08.5/0065.1/2001 and by an institutional grant by Fundacio´n Ramon Areces to CBMSO. R. Rial-Otero acknowledges the post-doctoral grant SFRH/BPD/23072/2005 from FCT (Science and Technical Foundation), Portugal. Financial support by FCT and FEDER under Projects POCI/QUI/55519/2004 and MetalControl N-1734 is also acknowledged. J.L. Capelo acknowledges the MALDI-TOF-MS Service of the Chemistry Department of the New University of Lisbon (http://www.dq.fct.unl.pt/eggebap/) for helpful assistance and valuable suggestions. The research findings reported are protected by international laws under patent request PORT No. 23848 of the INI, Instituto Nacional da Propiedade Industrial, Portugal. References [1] M. Baker, Nat. Biotechnol. 23 (2005) 297. [2] D.H. Chace, Chem. Rev. 101 (2001) 445. [3] P.A. Demirev, A.B. Feldman, J.S. Lin, Johns Hopkins APL Tech. Dig. 25 (2004) 27. [4] M.J. Stump, G. Black, A. Fox, K.F. Fox, C.E. Turick, M. Matthews, J. Sep. Sci. 28 (2005) 1642. [5] R.R. Drake, Y.P. Deng, E.E. Schwegler, S. Gravenstein, Expert Rev. Proteomics 2 (2005) 203. [6] Z.P. Yao, P.A. Demirev, C. Fenselau, Anal. Chem. 74 (2002) 2529. [7] W.J. Henzel, C. Watanabe, J.T. Stults, J. Am. Soc. Mass. Spectrom. 14 (2003) 931. [8] D.J. Pappin, P. Hojrup, A.J. Bleasby, Curr. Biol. 3 (1993) 327. [9] R.G. Krishna, F. Wold, in: R.H. Angeletti (Editor), Proteins, Analysis and Design, Academic Press, Oxford, UK, 1998 Chapter 2. [10] H. Neurath, in: R.J. Beynon, J.S. Bond (Editors), Proteolytic Enzymes: A Practical Approach, Oxford University Press, Oxford, UK, 1989 Chapter1. [11] J. Rosenfeld, J. Capdevielle, J.C. Guillemot, P. Ferrara, Anal. Biochem. 203 (1992) 173. [12] A.J. Link, J. Eng, D.M. Schieltz, E. Carmack, G.J. Mize, D.R. Morris, B.M. Garvik, J.R. Yates, Nat. Biotechnol. 17 (1999) 676. [13] Y.L.F. Hsieh, H.Q. Wang, C. Elicone, J. Mark, SA. Martin, F. Regnier, Anal. Chem. 68 (1996) 455. [14] A. Go¨rg, W. Weiss, M.J. Dunn, Proteomics 4 (2004) 3665. [15] L. Jiang, L. He, M. Fountoulakis, J. Chromatogr. A 1023 (2004) 317. [16] T. Rabilloud, Electrophoresis 19 (1998) 758.
Trends [17] B. Bjellqvist, K. Ek, P.G. Righetti, E. Gianazza, A. Gorg, R. Westermeier, W. Postel, J. Biochem. Biophys. Methods 6 (1982) 317. [18] W.M. Lauber, J.A. Carroll, D.R. Dufield, J.R. Kiesel, M.R. Radabaugh, J.P. Malone, Electrophoresis 22 (2001) 906. [19] L.D. Adams, K.M. Weaver, Appl. Theor. Electrophor. 1 (1990) 279. [20] M.J. Dunn, Methods Mol. Biol. 244 (2004) 339. [21] C.A. Retamal, P. Thiebaut, E.W. Alves, Anal. Biochem 268 (1999) 15. [22] M. Wilm, A. Shevchenko, T. Houthaeve, S. Breit, L. Schweigerer, T. Fotsis, M. Mann, Nature (London) 379 (1996) 466. [23] P. Kumarathasan, S. Mohottalage, P. Goegan, R. Vincent, Anal. Biochem. 346 (2005) 85. [24] F. Gharahdaghi, C.R. Weinberg, D.A. Meagher, B.S. Imai, S.M. Mische, Electrophoresis 20 (1999) 601. [25] F. Hillenkamp, M. Karas, R.C. Beavis, B.T. Chait, Anal. Chem. 63 (1991) A1193. [26] P.A. Nielsen, J.V. Olsen, A.V. Podtelejnikov, J.R. Andersen, M. Mann, J.R. Wisniewski, Mol. Cell. Proteomics 4 (2005) 402. [27] M.P. Washburn, D. Wolters, J.R. Yates, Nat. Biotechnol. 19 (2001) 242. [28] C.C. Wu, J.R. Yates, Nat. Biotechnol. 21 (2003) 262. [29] J. Blonder, T.P. Conrads, L.R. Yu, A. Terunuma, G.M. Janini, H.J. Issaq, J.C. Vogel, T.D. Veenstra, Proteomics 4 (2004) 31. [30] A.R. Ross, P.J. Lee, D.L. Smith, J.I. Langridge, A.D. Whetton, S.J. Gaskell, Proteomics 2 (2002) 928. [31] R.G. Sadygov, D. Cociorva, J.R. Yates, Nat. Methods 1 (2004) 195. [32] J.R. Freije, P.P.M.F.A. Mulder, W. Werkman, L. Rieux, H.A.G. Niederlander, E. Verpoorte, R. Bischoff, J. Proteome Res. 4 (2005) 1805. [33] http://docs.appliedbiosystems.com/pebiodocs/80134401.pdf. [34] D. Craft, L. Li, Anal. Chem. 77 (2005) 2649. [35] G. Massolini, E. Calleri, J. Sep. Sci. 28 (2005) 7. [36] G.W. Slysz, D.C. Schriemer, Rapid Commun. Mass Spectrom. 17 (2003) 1044. [37] J. Havlis, H. Thomas, M. Sebela, A. Shevchenko, Anal. Chem. 75 (2003) 1300. [38] B.N. Pramanik, U.A. Mirza, Y.H. Ing, Y.H. Liu, P.L. Bartner, P.C. Weber, M.K. Bose, Protein Sci. 11 (2002) 2676. [39] H.F. Juan, S.C. Chang, H.C. Huang, S.T. Chen, Proteomics 5 (2005) 840. [40] W. Sun, F.X. Li, J. Wang, D.X. Zheng, Y.H. Gao, Mol. Cell. Proteomics 3 (2004) 1194. [41] D. Lo´pez-Ferrer, J.L. Capelo, J. Va´zquez, J. Proteome Res. 4 (2005) 1569. [42] M. Postema, A. Van Wamel, C.T. Lancee, N. De Jong, Ultrasound Med. Biol. 30 (2004) 827. [43] P. Bermejo, J.L. Capelo, A. Mota, Y. Madrid, C. Camara, Trends Anal. Chem. 23 (2004) 654. [44] Bandelin electronic, GmbH & Co KG, Heinrichstrasse 3-4, D-12207. Berlin, Germany, http://www.bandelin.com. [45] Hielsher Ultrasonics GmbH, Warthestrasse 21, 14513, Teltow, Germany, http://www.hielscher.com. [46] Matrical, Inc. 665 North River Point Blvd. Spokane, WA 99202, USA, http://www.matrical.com/SonicMan.htm. [47] D.J. Halls, J. Anal. At. Spectrom. 10 (1995) 169. [48] R.J. Arnold, P. Hrncirova, K. Annaiah, M.V. Novotny, J. Proteome Res. 3 (2004) 653. [49] G. Pohlentz, S. Kolbl, J. Peter-Katalinic, Proteomics 5 (2005) 1758. [50] A. Emmer, J. Roeraade, Anal. Chim. Acta 542 (2005) 137.
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Sample treatment for protein identification by mass spectrometry-based techniques D. Lo´pez-Ferrer, B. Can˜as, J. Va´zquez, C. Lodeiro, R. Rial-Otero, I. Moura, J.L. Capelo Rapid identification of proteins is of primary importance for the analytical community. Protein-biomarker discovery for medical diagnostics or pharmacological purposes is becoming one of the hottest research topics. Moreover, rapid identification of proteins can help unambiguous bacterial and virus detection. In addition, the fast identification of bacteria can be used to beat bioterrorism. As a consequence, new analytical methodologies have emerged recently with the aim of making protein analysis as fast and as confident as possible. In this article, we critically review the new trends in sample treatment for protein identification and comment on the prospects for the future in this promising analytical area. ª 2006 Elsevier Ltd. All rights reserved. Keywords: Biomarker; Protein; Rapid identification; Sample treatment Abbreviations: ALS, Acid-labile surfactant; BSA, Bovine serum albumin; BTRFE, Bovine apotransferrin; CE, Capillary electrophoresis; CID, Collisioninduced dissociation; DTT, Dithiothreitol; IAA, Iodoacetamide; 2DE, Two-dimensional electrophoresis; ESI, Electrospray ionization; HIFU, Highintensity focused ultrasound; hTf, Human transferrin; IEF, Isoelectric focusing; LC, Liquid chromatography; MALDI-TOF, Matrix-assisted laser desorption/ionization – time-of-flight; MAPED, Microwave-assisted protein enzymatic digestion; MS, Mass spectrometry; PAGE, Polyacrilamide gel electrophoresis; PFF, Peptide-fragment fingerprinting; PMF, Peptide-mass fingerprinting; Q-TOF, Quadrupole time-of-flight; RP, Reversed phase; SCX, Strong cation exchange; SDS, Sodium dodecyl sulphate; TCA, Trichloroacetic acid.
1. Introduction D. Lo´pez-Ferrer, J. Va´zquez Centro de Biologı´a Molecular ‘‘Severo Ochoa’’-CSIC, 28049, Cantoblanco, Madrid, Spain B. Can˜as Departamento de Quı´mica Analı´tica, Facultad de Quı´micas, Universidad Complutense de Madrid, Madrid 28040, Spain C. Lodeiro, R. Rial-Otero, I. Moura, J.L. Capelo* REQUIMTE, Departamento de Quı´mica, Faculdade de Ciencia e Tecnologia, Universidade Nova de Lisboa, Monte de Caparica, 2829-516, Caparica, Portugal
*
Corresponding author. Tel.: +351 212 949 649; Fax: +351 21 294 8550; E-mail: [email protected]
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The identification of proteins is becoming more important in the scientific community due to emerging issues critically significant to society. Many hereditary diseases, some types of cancer and common diseases, such as diabetes, can be differentiated on the basis of the expression of certain proteins, known as protein biomarkers. As a result, disease screening and medical diagnosis take advantage of mass spectrometry (MS)-based technological improvements that allow ultra-fast protein identification [1,2]. Fast identification of protein biomarkers to identify bacteria is helping to reduce morbidity and mortality significantly across the globe [3,4]. Furthermore, rapid bacterial assessment through protein identification will help civil and military defense organizations to
0165-9936/$ - see front matter ª 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2006.05.015
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beat bioterrorism [5], and recent literature suggests that protein identification can also be applied to assessing viruses [6]. The main limiting steps for rapid protein identification are (i) the number of protein entries in database; and, (ii) sample treatment. The second limitation is due to the handling protocols commonly used for protein identification. Traditionally, these protocols are tedious, with many single steps. In addition, from four hours to overnight is the time usually required for enzymatic digestion for protein identification. Most improvements reported in literature in the past five years have been devoted to enhancing the throughput of sample treatments. The main aims of this article are: (i) to review advances in sample treatment for fast protein identification; (ii) to discuss the parameters affecting each new methodology along with their advantages and drawbacks; (iii) to show current applications of protein identification; and, (iv) to comment future prospects.
2. Enzymatic digestion of proteins Protein isolation or purification is the first step in protein identification. The second step is specific cleavage of the peptide bonds in a protein to produce peptides, which is mainly done using enzymes, obtaining an enzymespecific pool of peptides; this procedure is known as protein digestion. The characteristic pool of peptides produced is then analyzed by MS-based techniques, such as matrix-assisted laser desorption/ionization time-offlight MS (MALDI-TOF-MS) or liquid chromatographyMS (LC-MS), to obtain accurate measurements of their masses. In most cases, this set of masses is enough for unambiguous identification of the protein. This methodology is known as peptide-mass fingerprint, PMF [7], which involves comparing the experimental masses from the peptides produced by the digested protein, and those produced by in silico, theoretical, digestion of all the proteins in a particular database. Special search programs [8], known as search engines, are used for this purpose. Here it is found the Achilles Heel for fast protein identification: the sequence of the protein to be identified must be included in the database; otherwise, PMF fails to identify the protein. 2.1. Types of enzymes: intended effects As stated above, peptides are produced from proteins through enzymatic digestion. Nevertheless, this is not the only way to digest proteins, since special chemical reagents, such as cyanogen bromide [9], can be also used. The choice between chemical or enzymatic protein
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cleavage will depend on several factors (e.g., the type of protein, its sequence and the purpose of the analysis). Enzymatic digestion presents several advantages for PMF, mainly due to the size of the peptides produced, which is very compatible with their analysis by MS. The enzymes commonly used for protein digestion comprise a group of proteins called proteases, which are hydrolytic enzymes that act on the amide bonds at specific positions in the protein under digestion. There are different types of proteases; the most frequently cited in the literature belong to one of the following groups [10]: (i) serine; (ii) aspartic; (iii) cysteine; or, (iv) metalloproteases. Differences among proteases account for (i) the mechanism of protein cleavage, and (ii) conditions for optimum performance. Concerning mechanisms of protein cleavage, when the aim of the analysis is protein identification, trypsin, a serine protease, is the most widely used enzyme to perform protein digestion, since it presents the following advantages: (i) it cleaves exclusively after arginine or lysine residues, which are present in proteins at the approximate rate of one residue per every 10–12 amino-acids. Peptides produced have an average size of 800–2000 Da, very adequate for MS analysis; and, (ii) peptides resulting from trypsin digestion produce very homogeneous fragmentation under collision-induced dissociation (CID), a special methodology in MS, so fragmentation spectra of only one tryptic peptide may give enough information to identify a protein, if it is included in the database. As for conditions for performance, for most enzymes, activity is temperature dependent until a maximum is reached, when the enzyme is no longer stable. Furthermore, enzymes, as many other proteins, are stable only over a limited range of pH; outside this range, changes in the charges of ionizable amino-acid residues may result in modification of the tertiary structure of the protein and eventually lead to enzyme denaturation and inactivation. There is a pH value at which the activity of the enzyme reaches a maximum or a plateau, so protein digestion is usually performed in a buffered medium. Enzyme concentration also affects the process, so it needs to be optimized. Three different strategies can be used for protein digestion using enzymes, as summarized in Fig. 1. In the first procedure, protocol A, proteins initially isolated by SDS-PAGE or two-dimensional electrophoresis (2DE), are digested in situ, inside the gel structure. This methodology is known as in-gel protein digestion [11]. In the second approach, protocol B, cell extracts, comprising protein mixtures with different degrees of complexity, are enzymatically digested, sometimes without previous purification. This methodology is called in-solution digestion [12]. This procedure has several advantages (e.g., compatibility of protein identifications carried out at the same time). In the third strategy, protocol C, the http://www.elsevier.com/locate/trac
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solution containing a protein mixture is passed through a column filled with a chromatographic packing containing an immobilized enzyme. This is the in-column digestion methodology and is very useful for simple mixtures of proteins in solution [13]. Using the in-column approach, it is possible to couple protein digestion, peptide separation and MS analysis, so facilitating the complete analysis on-line. Table 1 shows the advantages and drawbacks of these three sample treatments, which we treat in detail below.
Cell lysis
Protein Cell Extract
Protocol A
Protocol B
Protocol C
Protein denaturation
Protein precipitation
HPLC protein separation
1D or 2D-SDS-PAGE protein separation
Protein denaturation
Isolated Proteins
Isolated Proteins
Reduction & alkylation Steps
Enzyme digestion (4-12 hours, 37 ºC)
Pass through immobilized trypsin cartridges
Spot excision
Enzyme digestion (4-12 hours, 37 ºC)
Stop hydrolysis (acid addition) Peptide Pool
Stop hydrolysis (acid addition)
Complex peptide pool
Peptide Pool 2D-HPLC peptide separation
HPLC peptide separation
Mass Spectrometry Analysis
Protein Identification
Figure 1. Enzymatic protein-digestion protocols. Protocol A corresponds to the classical protein analysis, in which proteins are initially separated according to their isoelectric point and then according to their MW. Protocol B corresponds to the typical gel-free approach, in which proteins are digested prior to any separation step and then the whole peptide pool is separated using multi-dimensional chromatography in tandem with mass spectrometry. Protocol C represents the new trends in protein digestion using immobilized trypsin. Firstly, proteins can be separated using different chromatography steps, and are then digested in-column, and, finally, the peptide pool is separated and analyzed.
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2.2. In-gel protein digestion This is by far the most tedious and time-consuming approach, and most dedicated advances in recent literature are devoted to speeding up this methodology, as we will show later. Fig. 2 shows a general scheme for this sample treatment, in which the common steps for in-gel protein digestion are presented in conjunction with their intended effects. As can be seen, there are many steps that must be followed to digest a protein correctly. First, proteins must be separated and purified. The most frequent separation strategy used for complex protein mixtures is one-dimensional electrophoresis or 2DE, as previously reviewed [14]. Depending on the sample, it may be necessary to concentrate and eliminate interfering substances before electrophoretic separation. In such a case, it is good practice to precipitate protein extracts with a mixture of trichloroacetic acid (TCA)/ acetone [15]. To ensure efficient, reproducible separation in the gel, protein extracts must previously be denatured in urea/thiourea at high concentrations, 8–9 M. In addition, thiourea increases the solubility of hydrophobic proteins [16]. To run the first dimension of 2DE, samples are applied to isoelectric focusing (IEF) gel strips containing ampholites, substances with different pKa values that create a pH gradient along the gel. Nowadays, gel strips for the first dimension are prepared with immobilized ampholites, in the form of acrylamide derivatives, called immobilines [17], which dramatically increase reproducibility. Once the first dimension is completed, and to prevent further re-oxidation, protein-cystine residues must be reduced using dithiothreitol (DTT) and the resulting cysteines blocked with iodoacetamide (IAA). After focusing, IEF strips are soaked in SDS to prepare proteins for separation by molecular masses (MW) in the second dimension. When the separation is complete, proteins are visualized in the gel by staining with Coomassie Blue, fluorescent dyes, or with MS-compatible silver nitrate [18]. The result of the 2DE separation procedure is shown in protocol A (Fig. 1), in which each dark spot corresponds, theoretically, to a protein.
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Table 1. Analytical parameters for enzymatic protein digestion Variable
In-gel digestion
In-solution digestion
In-column digestion
Treatment time Handling Sample throughput On-line application Protein-digestion yield Robustness Enzyme-to-protein ratio allowed Cost
up to 12 h high low no low low low low
up to 12 h medium medium yes medium high very low low
up to 10 min. low high yes high medium medium medium
Further treatment may require intact proteins to be eluted from the gel. Proteins may be extracted from the gel by different techniques (e.g., (i) passive elution [19], (ii) electroelution [20], or (iii) the ultrasonic-assisted elution [21]). Once eluted, proteins are digested in solution and then analyzed by PMF. Nevertheless, protein elution from gels is troublesome, being protein dependent and, generally, with very low recovery yields. This is why most researchers in the field of protein identification prefer to digest proteins in situ, inside the gel, without previous elution. This procedure is based on the peptides are most easily eluted from gels than the original, undigested proteins, so the problems associated with protein elution from gels are overcome. As a result, robust protocols have been developed for in-gel digestion [22]. In addition, automated protein digestion is available in many proteomic facilities. To proceed with in-gel digestion of proteins, separate protein bands or spots are cut from the gel and de-stained, following procedures that depend on the staining procedure. There are protocols for removing Coomassie-blue [23] or silver [24] from pieces of gel. Once de-stained, and to ensure adequate digestion of the protein inside the gel, the following parameters must be optimized: (i) accessibility of the enzyme to the cleavable bonds may sometimes be low, because proteins are trapped inside the gel structure, so it is very important to maintain an adequate enzyme/substrate ratio to maximize in-gel digestion, this ratio being much higher than that needed for in-solution digestion; (ii) solvent composition inside the gel must be strictly controlled, in order to achieve a good digestion yield; for this purpose, gel slides must first be dehydrated using acetonitrile and dried in a speedvacuum, the objective of this treatment being to facilitate in-gel penetration of the solution containing the enzyme, which is added after gel drying; and, (iii) the pH and temperature of the solution must be controlled; otherwise, enzyme efficiency can be lowered.
From a few hours to overnight incubation at 30C or 37C is usually enough for complete protein digestion. Finally, the supernatant containing the peptides is acidified to stop the digestion process and subjected to MS analysis, generally using a MALDI-TOF instrument [25] for protein identification by PMF. The relation between the amino-acidic sequence found experimentally and the theoretical sequence of the assigned protein, expressed as percentage, is known as the sequence coverage of the PMF, and is crucial for trust in protein identification. The in-gel digestion protocol presents several drawbacks, namely (i) trapping protein substrates in the gel makes some peptide bonds inaccessible to the enzyme, and (ii) not all the peptides produced during digestion can diffuse freely from the gel. This explains the difference in results between in-gel and in-solution digestion. Table 2 shows selected examples of in-gel protein digestion. 2.3. In-solution protein digestion Shotgun proteomics, initially developed by Link et al. [12], has become very popular in the past few years. It is based in whole-extract protein digestion in solution. This procedure was extensively used in the analysis of isolated proteins, but is now applied to complex protein mixtures. Protein composition and their particular chemical and physical properties are very diverse. As a result, the degree of protein digestion and the type and quantity of peptides obtained differ greatly from one protein to another. When working with this procedure, it is important to keep in mind the following: (i) more abundant proteins are usually easier to identify; (ii) to facilitate identification of minor proteins, some fractionation (e.g., organelle purification) is needed; and, (iii) variation in the yield of digestion from sample to sample can occur because of (1) heterogeneity in the matrix, (2) the quantity and number of proteins, and (3) physical and chemical properties of the proteins.
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Classical Protein Analysis Treatment for 1D Gels: total time 20-24 h.
Purification
5µl of protein + 5µl of Sample buffer for SDS-PAGE prepared as follows: tris-base 0,5M at pH=6,6–6,8 (5ml) +10% SDS (8ml) + β -mercaptoethanol (1ml) + glycerol (2ml) + bromophenol blue (4mg) + H2O till 20ml of total volume
Boiling in water bath for 5 min
Reduction of disulfide bonds (βmercaptoethanol) Visualize the sample electrophoretic front (Bromophenol Blue) Optimize pH for trypsin activity (TrisBase 0,5M) Remove protein charges (SDS)
Protein denaturation
Electrophoresis (60/70 min) at constant voltage of 120V and 400mA
Protein separation and purification
Staining with coomasie blue R-250 (1g) + glacial acetic acid (15ml) + methanol (90ml) + H2O till 200ml of total volume
Visualize the proteins bands through staining
De-staining with a decolouring solution prepared with: glacial acetic acid (75ml) + methanol (450ml) + H2O till 1000 ml of total volume
Remove the excess of colouring solution
Cut off protein spots and slight slicing the gel with the scalpel
Wash with water (3 x 100µl) and acetonitrile (3 x 100µl)
Treated with DTT (40 µl, 25 min) and IAA (30µl, 35 min)
Sample Preparation
Intended Effect
Wash with water (3 x 100µl) and acetonitrile (3 x 100µl)
Dry in a dried speed- vacuum
Add trypsin (15 µl) and left in a ice bath for 1 h
Facilitate trypsin penetration into the gel
Removal interferences
Reduction and alkylation di-sulphide bonds
Removal interferences
Dehydrate the gel
Penetration of the enzyme into the gel without digestion
Add NH4HCO3 buffer 12,5mM (10µl)
Maintain pH during incubation time
Incubate at 37ºC for 4 h or overnight. (HIFU sonication for 2 minutes)
Digestion of proteins into peptides
Add formic acid 0,3% (15µl)
Stop trypsin activity
Figure 2. Detailed description of an in-gel protein digestion protocol.
As a general rule, to overcome these drawbacks, protein extracts are: (a) first precipitated using cold acetone or TCA; (b) after precipitation, and in order to make the proteins more accessible to the action of the enzymes, proteins are re-suspended in ammonium bicarbonate buffer supplemented with 8 M urea or another chaotropic agent (e.g., guanidine hydrochloride) to break intra-molecular forces and denature proteins; and, finally, 1000
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(c) to prevent protein renaturation before enzymatic attack, reduction and alkylation of protein disulfide bridges are commonly done using DTT and IAA, although other reagents are also available for the same purposes. As mentioned above, trypsin is the enzyme chosen for protein digestion because of its specificity [22]. However, some in-solution protocols [26,27] make use of two digestion steps, first Lysine-C (Lys-C) and then trypsin, because Lys-C fully preserves activity in the presence of
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Table 2. Selected sample treatments for enzymatic protein digestion Analytical task In-gel protein digestion Identification of proteins from 2D-PAGE with a new acid-labile surfactant
Comparison of protein-precipitation methods for sample preparation prior to proteomic analysis
Protein in-gel digestion for proteome characterization
Comparison of different staining procedures for MS
In-solution protein digestion A largely unbiased method for rapid, large-scale proteome analysis
Proteomic mapping of brain plasma-membrane proteins
A simple, rapid method for characterizing hydrophobic integral membrane proteins.
Fast proteolytic digestion coupled with organelle enrichment for proteomic analysis of rat liver.
In-column protein digestion Ion-electrospray capillary proteolysis
Analytical techniques/comments
Ref.
MALDI-TOF-MS / Results indicate that substituting SDS by ALS, the numbers of peptides detected by MALDI-TOF-MS are significantly increased, especially for proteins of low abundance Spot identity of gel-slides was done by similarity with previously scanned gels / Four protein-precipitation methods were compared: (i) trichloroacetic acid, (ii) acetone, (iii) chloroform/methanol, and (iv) ammonium sulfate. Precipitation of proteins with TCA and acetone and ultra-filtration result in an efficient sample concentration and desalting for proteomic analysis MALDI-TOF-MS / A study was conducted to obtain a fast, reliable approach to enhance in-gel protein digestion and high peptide recovery. MOWSE score obtained was twice that of the standard protocol MALDI-TOF-MS and nano-LC-MS2(Q-TOF) / The following staining procedures were compared: (i) colloidal Coomassie blue, (ii) Daiichi Silver, (iii) SYPRO Orange, (iv) SYPRO Red, (v) SYPRO Tangerine, and (vi) SYPRO Ruby. Best results with SYPRO Red and Orange
[30]
Multidimensional LC-MS2 / A total of 1484 proteins were detected and identified (proteome of Saccharomyces cerevisiae strain BJ5460). 131 proteins with three or more predicted transmembrane domains. Complex peptide mixtures were loaded separately onto a biphasic microcapillary column packed with strong cation exchange (SCX) and reverse-phase (RP) materials LC-MS2 (Q-TOF) and LC-MS2 (Ion-Trap-FT) / A novel method for extraction and fractionation of membranes, onmembrane extraction followed by on-membrane digestion and diagonal separation of peptides. 862 proteins from 150 mg of mouse brain cortex and 1685 proteins from 15 mg of hippocampus were identified Micro-capillary LC-MS2 (lLC-MS2)/A detergent and cyanogen bromide-free method for integral membrane proteomics was applied to Halobacterium purple membranes and the human epidermal membrane proteome. From human epidermal sheets, a total of 117 proteins were identified LC-MS2(Ion-Trap) / The use of an acid-labile surfactant (ALS) as an alternative to urea denaturation was demonstrated by comparison of the same sample treated with (i) both organelle enrichment and whole-cell lysate or (ii) reduction, alkylation, and urea denaturation. Number of peptides obtained: 694 for urea, 674 for ALS; number of proteins 225 for urea, 229 for ALS
Q-TOF-MS (Positive ion mode (ESI (+)). / The ionization source was set at 80C. 10 pmol/lL of BSA or BTRFE dissolved in (5–10 mM NH4HCO3 + 10–20% methanol) plus final Trypsin or endoproteinase Glu-C concentration of 0.02–0.4 pmol/lL, respectively. The mixture was transferred to the electrospray capillary without further treatment. Sequence coverage: >40% with trypsin and 100% with Glu-C. Validation with elephant milk protein preparation: five proteins were correctly identified
[15]
[23]
[18]
[27]
[26]
[29]
[48]
[49]
(continued on next page)
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Table 2 (continued) Analytical task
Analytical techniques/comments
Ref.
Chip-based nanovials proteolytic digestion
CE; MALDI-TOF / Two different protocols are described for enzyme (trypsin, chymotrypsin) immobilization in nanovials. Nanoliter-scale enzymatic digestion. Digestion time 3–4 h at 37C. Chip re-utilization impossible MALDI-TOF-MS / Three water mixtures (methanol, isopropanol, acetonitrile) were used for digestion of human transferrin (hTf). Digestions with methanol produced fewer missed cleavages, whilst, with acetonitrile, they produced most peptide and intense mass spectra. Digestion of low nanomolar myoglobin was OK LC-ESI-MS2 / Steps: (i) capture the proteins on the column packing material; (ii) fill column with enzyme for protein digestion; (iii) wash column: elution of enzyme excess; (iv) elute peptide fragments. Total time: 80 min., each cycle. 3 HPLC pumps, 2 C8 columns and 2 micro-digestion columns
[50]
On-column digestion in aqueous-organic solvents
Digestion with immobilized proteins
8 M urea, the accessibility of this enzyme to peptide bonds being greater than trypsin; this activity is possible only after a urea dilution to 2 M. Protein digestion is usually performed for 12–24 hours, due to protein heterogeneity in samples. Finally, the reaction is stopped by adding an acid, such as trifluoroacetic or formic, to a pH of 2–3. Then, the digest can be directly analyzed by 2DE-LC-MS2. In some cases, lyophilization or evaporation in a speed vacuum can be used for (i) further sample concentration or (ii) sample storage until analysis; in this case, temperature preservation is needed at, or even below, 20C. Hydrophobic membrane proteins, with low solubility in water, represent a special case. These proteins play important roles in various cellular processes, such as signal transduction and cell adhesion. Cyanogen bromide at high concentrations of acid [28] or tryptic digestion at high concentrations of methanol [29] have been reported in the literature to handle membrane proteins successfully. A promising methodology for membrane proteins uses acid-labile surfactants (ALSs) [30]. Furthermore, using ALS steps (e.g., urea denaturation of proteins), disulfide reduction/alkylation may be eliminated. In addition, proteolytic digests performed in the presence of ALS are easy to recover and do not need desalting steps for further chromatography and MS analysis. Table 2 shows selected examples of in-solution protein digestion. The complex mixture of peptides produced by the insolution digestion procedure is separated using multi-dimensional LC, coupled to electrospary ionization (ESI)-MS2 [12]. Peptides are identified by their fragmentation spectra, in a similar way to PMF, but using specialized software that compares the experimental fragmentation pattern of a peptide with that generated in silico from all the proteins in a database. This procedure is known as peptide-fragment fingerprinting (PFF) [31]. 1002
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[36]
[34]
2.4. In-column protein digestion Usually, tryptic protein digestion is done either in gel or in solution, as described above, although these methodologies have several drawbacks [32]: (i) long digestion time (typically >5 h); (ii) low trypsin-to-substrate ratios are allowed to avoid the production of interfering peptides from trypsin autolysis; (iii) low digestion yields obtain when the substrate concentration is in the low-micromolar range; (iv) time-consuming handling; and, (v) peptide losses by adsorption can occur. To overcome these problems, proteolytic digestion may be performed in solid supports containing immobilized enzymes. This procedure, called in-column digestion, has the following advantages: (i) lower digestion times; (ii) increased digestion ratios; and, (iii) incorporation into multi-dimensional separation procedures for automated proteomics. Several companies provide cartridges with highdensity immobilized trypsin (e.g., the Poroszyme cartridge that can be used into integrated systems including MS detection for peptide identification [33,34]). However, high cost, limited catalytic activity and autolysis are still limitations that need to be overcome. In this methodology: (i) multi-dimensional chromatography is used to purify proteins from mixtures; (ii) the separated proteins are then digested by passing them through immobilized trypsin cartridges; and, (iii) the peptides formed are separated by reverse phase HPLC. As a result, integrated protein identification is possible. Trypsin can be immobilized chemically, trapped or physically adsorbed. The packing material used for enzyme immobilization is usually made of silica or
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polymeric material, and is packed as particulate or as monolithic columns. Monolithic columns are preferred because of their low cost and relatively little resistance to solvent flow, and that allows fast analysis and higher throughput. These properties are due to their open, rigid structure. The properties of the material used for enzyme immobilization, such as shape or porosity, affect the performance of the digestion reactor. The review based on chemical reactors for on-line systems by Massolini and Calleri [35] is highly recommended. Freije et al. [32] reported enhancement in proteolysis activity when trypsin reactions involved chemically modified (acetylated) trypsin. In addition, they found fewer interfering trypsin autolysis products. The use of water mixed with organic solvents may help in incolumn digestion of resistant proteins, as was demonstrated by Slysz and Schriemer [36]. Furthermore, a complex system (Fig. 3, coupling three HPLC pumps, two digestion columns, two separation columns and three injection valves) has been described in the literature [34]. This analytical approach may be necessary for the following reasons: (i) to prevent losses of hydrophilic peptides after their elution from the trypsin cartridge; (ii) to trap peptides in a capillary column before separation by one-dimensional or two-dimensional chromatography; and, (iii) to avoid detrimental signal suppression in the ESI process by salts by installing a desalting column. Table 2 shows selected examples of in-column digestion.
3. Accelerating protein digestion As stated above, the most recent research reported in the literature has been devoted to the acceleration of the enzymatic digestion process, since it is the most timeconsuming step in identifying proteins. First trials were done taking advantage of basic concepts in enzymology, such as the role of enzyme concentration and the influence of temperature on the rate of the reaction. Using these concepts, a fully enzymatic in-gel protein digestion was achieved in less than 1 hour [37]. There may also be improvements in kinetics, when the digestion is subjected to microwave energy. Recently, an ultra-fast method was developed using high-intensity, focused ultrasound (HIFU). We will comment on both of these methodologies below. 3.1. Microwaves Microwave-assisted protein enzymatic digestion (MAPED) is useful for in-solution [38] or in-gel [39] digestion and may be also applied to complex protein mixtures [40]. Using this methodology, digestion times required to get a complete digestion are up to 20 min. Low irradiation power is used; below 30% of the total nominal power of the microwave oven is recommended; otherwise, temperature control becomes difficult. Critical parameters to be optimized in this procedure are (i) irradiation time, (ii) temperature, and (iii) power. 3.2. Ultrasound The application of HIFU to sample treatment for fast protein identification was first developed by Lo´pez-Ferrer
Digestion Column Colum B AS-HPLC -HPLC 1 system
2 3
1
Separation Column 2
4 10
5 9
8
7
Waste
2
1
6
3 6
AS-HPLC 2 system
Digestion Column A
1 5
Q1
2 3
Ion Prec . C. Cell
4
4
Q2
Q3 LIT
5 6
HPLC pump 3
Waste Mass Spectrometer
Separation Column Colum 1
Figure 3. An on-line protein digestion system. comprising three HPLC pumps (two gradient and one isocratic), three switching valves, and four columns (two used for protein digestion and two other for peptide separation). The whole system is connected on line to a mass spectrometer. To increase throughput of the analysis, each gradient HPLC pump is connected to one digestion column and one separation column through switching valves. These are synchronized to separate (separation-column 1) the digested peptides coming from digestion-column A using one of the gradient HPLC systems. In the meantime, the second gradient system delivers a protein mixture to digestion-column B, while separation-column 2 is being conditioned with the solvent delivered by the isocratic pump. (Adapted from [34], courtesy of The American Chemical Society).
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et al. [41]. Fig. 4 shows HIFU/enzymatic digestion in progress along with the main characteristics. In in-solution protein digestion, the procedure entails sonication with HIFU of small volumes of sample, typically 20–50 lL while the trypsin digestion proceeds. The HIFU treatment probably boosts enzyme-substrate kinetics. The procedure is completed in less than 15 s. In in-gel protein digestion, slides of gel containing protein can be subjected to the same procedure: once excised, the gel piece is introduced in an Eppendorf cup and a small amount of a buffer containing the enzyme is added. The liquid jets produced by the HIFU act as microsyringes [42], delivering the enzyme into the gel and making the protein digestion faster than with any other methodology for in-gel protein digestion: 15–30 s. In addition, the amount of enzyme necessary is three times less than that needed without ultrasonic energy. The mechanical erosion of the gel surface caused by the cavitation associated with ultrasonication enhances peptide release from the gel. This finding deserves more research and is currently being developed in our laboratory. Ultrasonic probes specially developed for small volumes, 10–100 lL, must be used when only minute amounts of protein sample are available. In addition, the use of small sample volumes helps (i) to concentrate the sample, (ii) to reduce time of treatment when the speed vacuum or lyophilization is expected to be used, and (iii) to reduce the amount of enzyme trypsin used. The following parameters must be addressed to optimize performance of this methodology: (a) sonication time; (b) sonication power; (c) sonication amplitude;
1.5 or 0.5 ml reaction cup Ultrasonic probe Cavitation effects
Variables: (i) (ii) (iii) (iv) (v) (vi) ((vii))
Gel slide stained with comassie blue
Probe diameter Sample volume Sonication time Sonication amplitude Enzyme concentration Temperature Sonication power
Figure 4. Enzymatic in-gel protein digestion accelerated by highintensity focused ultrasound (HIFU).
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(d) sample volume; (e) enzyme-to-substrate ratio; (f) probe diameter; and, (g) temperature.
4. Future prospects We expect more research on accelerating chemical reactions using either microwave or ultrasonic energy. As far as microwave energy is concerned, we expect more research to be devoted to the domestic microwave oven, since these devices have not yet not been tried, probably because temperature control is difficult. However, domestic microwave ovens are very cheap, and on-line digestion procedures could be developed so that temperature control can be minimal [43]. We also expect more literature to report on-line procedures in conjunction with microwave energy. Regarding ultrasound, the following research is needed: (i) comparison of sonication probes from different manufacturers in order to assess the robustness of the procedure; (ii) assessment of new ultrasonic devices, such as (a) glass sonication probes [44], (b) sonication baths with different working wavelengths [44], or (c) new powerful sonoreactors [45]; (iii) applicability of the procedure to the recently developed multi-probe sonicators [46], since a minimum of 12 samples can be treated at once and protein digestion can be performed in seconds, thus allowing easy implementation of this procedure in robotic platforms for enzymatic digestion. The possibilities of high sample throughput along with its easy implementation in robotic devices gives this ultrasonic procedure an excellent future in the biotechnology industry and in proteome research. There is one other consideration: the enhancement of enzymatic protein digestion by ultrasonication meets the requirements of analytical minimalism, as proposed by Halls [47]: (1) low cost; (2) low sample requirements; (3) low reagent consumption; and, (4) low waste production.
5. Conclusions Protein identification is an issue of primary importance for the analytical community, its applications covering research areas as diverse as diagnosis of disease and defense against bioterrorism, and demanding the presence of analytical chemists. Improvements in sample treatment achieved using microwave energy or HIFU have changed the methodologies in such a way that protein identification can be
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done in less than 12 h, avoiding previous slow, tedious procedures. We expect protein digestion to be enhanced in the immediate future so that the limiting step in protein identification will be the MS technique chosen for protein analysis rather than the sample treatment. Acknowledgements We thank Antonio Ramos-Ferna´ndez for helpful advice and discussion. This work was supported by grants CICYT BIO2003-01805 and CAM 08.5/0065.1/2001 and by an institutional grant by Fundacio´n Ramon Areces to CBMSO. R. Rial-Otero acknowledges the post-doctoral grant SFRH/BPD/23072/2005 from FCT (Science and Technical Foundation), Portugal. Financial support by FCT and FEDER under Projects POCI/QUI/55519/2004 and MetalControl N-1734 is also acknowledged. J.L. Capelo acknowledges the MALDI-TOF-MS Service of the Chemistry Department of the New University of Lisbon (http://www.dq.fct.unl.pt/eggebap/) for helpful assistance and valuable suggestions. The research findings reported are protected by international laws under patent request PORT No. 23848 of the INI, Instituto Nacional da Propiedade Industrial, Portugal. References [1] M. Baker, Nat. Biotechnol. 23 (2005) 297. [2] D.H. Chace, Chem. Rev. 101 (2001) 445. [3] P.A. Demirev, A.B. Feldman, J.S. Lin, Johns Hopkins APL Tech. Dig. 25 (2004) 27. [4] M.J. Stump, G. Black, A. Fox, K.F. Fox, C.E. Turick, M. Matthews, J. Sep. Sci. 28 (2005) 1642. [5] R.R. Drake, Y.P. Deng, E.E. Schwegler, S. Gravenstein, Expert Rev. Proteomics 2 (2005) 203. [6] Z.P. Yao, P.A. Demirev, C. Fenselau, Anal. Chem. 74 (2002) 2529. [7] W.J. Henzel, C. Watanabe, J.T. Stults, J. Am. Soc. Mass. Spectrom. 14 (2003) 931. [8] D.J. Pappin, P. Hojrup, A.J. Bleasby, Curr. Biol. 3 (1993) 327. [9] R.G. Krishna, F. Wold, in: R.H. Angeletti (Editor), Proteins, Analysis and Design, Academic Press, Oxford, UK, 1998 Chapter 2. [10] H. Neurath, in: R.J. Beynon, J.S. Bond (Editors), Proteolytic Enzymes: A Practical Approach, Oxford University Press, Oxford, UK, 1989 Chapter1. [11] J. Rosenfeld, J. Capdevielle, J.C. Guillemot, P. Ferrara, Anal. Biochem. 203 (1992) 173. [12] A.J. Link, J. Eng, D.M. Schieltz, E. Carmack, G.J. Mize, D.R. Morris, B.M. Garvik, J.R. Yates, Nat. Biotechnol. 17 (1999) 676. [13] Y.L.F. Hsieh, H.Q. Wang, C. Elicone, J. Mark, SA. Martin, F. Regnier, Anal. Chem. 68 (1996) 455. [14] A. Go¨rg, W. Weiss, M.J. Dunn, Proteomics 4 (2004) 3665. [15] L. Jiang, L. He, M. Fountoulakis, J. Chromatogr. A 1023 (2004) 317. [16] T. Rabilloud, Electrophoresis 19 (1998) 758.
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