COFRADIC™: the Hubble telescope of proteomics

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COFRADIC™: the Hubble telescope of proteomics Kris Gevaert and Joël Vandekerckhove COFRADIC™ is a diagonal chromatographic technique designed to select peptides from complex mixtures. It consists of two consecutive identical chromatographic separations, with a modification step targeted to a subset of peptides between the two separations. The modified peptides obtain different chromatographic properties and segregate from the bulk of unaltered peptides in the second run. The final analysis is limited to the sorted peptides, thereby reducing the complexity but keeping all of the characteristics of the proteome. Kris Gevaert* Joël Vandekerckhove Department of Medical Protein Research Flanders Interuniversity Institute for Biotechnology Faculty of Medicine and Health Sciences Ghent University A. Baertsoenkaai 3 B9000 Ghent, Belgium *e-mail: [email protected]

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ganisms have become available in recent years, creating new perspectives for the global analysis of proteins. The proteome was originally studied by 2D polyacrylamide gel electrophoresis (2DPAGE) [1,2], and recent improvements in biological mass spectrometry (MS) combined with database searches have facilitated the fast identification of proteins separated on 2D gels [3]. Opposite to this gel-based approach stand peptide-based approaches, where the proteome is digested directly and the resultant peptides, identified by tandem MS (MS/MS) analysis, serve as signatures for their parent proteins. Because the latter methods do not use gels but rely predominantly on chromatographic technology, they are referred to collectively as ‘non-gel proteomics’. Greater coverage of a proteome is often obtained with non-gel techniques; on the whole, however, the two methods (gel and non-gel) are complementary. Two general approaches to non-gel methods can be distinguished. In the first, peptides are directly passed over orthogonal chromatographic systems in an attempt to obtain maximal separation before delivering them to the mass spectrometer [4–7]. In the second, the complexity of the mixture is reduced by first selecting a subset of peptides that are highly representative of the

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original proteins and then restricting the analyses to this subset [8–16].The combined fractional diagonal chromatography approach or COFRADIC [17–19] can be assigned to the latter type of non-gel approach. Unlike procedures that select signature peptides by affinity chromatography, however, COFRADIC is based on the principle of diagonal reversed-phase chromatography [20] and, as such, is extremely versatile in sorting different classes of peptides, thereby enabling targeted analysis and guaranteeing broad protein coverage. In this technical focus article, we describe examples of COFRADIC-based proteome analyses that are based on selecting methionyl, cysteinyl and amino (N)-terminal peptides and show that the technique can be used for the global analysis of phosphopeptides. What applications does COFRADIC™ have? As mentioned above, in the COFRADIC technique a specific class of peptides is isolated from a highly complex mixture. The only prerequisite is that these peptides contain at least one functional group that distinguishes them from all other peptides and that can be specifically and quantitatively modified by a chemical or enzymatic reaction.This modification step is performed between two consecutive identical chromatographic separations. If the chromatographic properties are altered sufficiently, the modified peptides will show different retention times when re-separated, whereas unaltered peptides will elute at the same position in the second separation.Thus, the modified peptides segregate from the unaltered ones and can be specifically collected for further analysis (Figure 1). In theory, COFRADIC can be applied to the isolation of any compound or family of compounds, provided that they can be specifically and quantitatively modified. Specificity is important because side reactions can induce the selection of

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Figure 1. Principle of COFRADIC for isolating representative peptides from a complex peptide mixture. In the primary run (top), a peptide mixture is separated on a reversed-phase high performance liquid chromatography column. The fractions are then subjected to chemical or enzymatic modification to alter the column retention properties of a class of peptide present in each primary fraction. In the secondary run (bottom), these modified primary fractions are reloaded on the same column and the peptides are separated under identical conditions. The modified peptides shift out of the original collection interval and can be specifically isolated for further analysis. The modified peptides either elute sooner (hydrophilic shift, −δ) or later (hydrophobic shift, +δ) than the unmodified peptides.

undesirable components, which complicates the subsequent analysis. So far, COFRADIC has been used to isolate representative peptides, including methionyl [17], cysteinyl [19] and N-terminal [18] peptides, from trypsin digests of a proteome. The N-terminal peptides are particularly useful because every

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protein is represented by only one peptide, thereby reducing a sample’s complexity to the highest degree. Applications of COFRADIC are not limited to peptides containing rare amino acids; in fact, all posttranslationally modified peptides that can be altered specifically fall into the application range. Below we illustrate this versatile characteristic of COFRADIC by the analysis of phosphopeptides and a discussion of forthcoming applications. How does COFRADIC™ work? The basic principle of COFRADIC is derived from the diagonal electrophoresis technique, which was originally published in 1966 [21]. The term ‘diagonal chromatography’ was subsequently coined when the procedure was carried out with reversedphase or ion-exchange chromatography instead of paper electrophoresis [20]. Whereas conventional diagonal chromatography is applicable to relatively simple peptide mixtures, COFRADIC is designed to handle highly complex mixtures. In the primary run the peptides are fractionated. Several primary fractions are then combined to reduce the number of secondary runs. Fractions are combined such that shifted peptides from one fraction will not overlap with unaltered peptides from other primary fractions. In this way, the total number of secondary runs can be reduced by at least a factor of 4. Under carefully controlled chromatographic conditions, a peptide is generally recovered in 1–2 secondary fractions, guaranteeing high sensitivity. Isolation of peptides containing methionine In addition to being a rare amino acid, methionine is well distributed in the proteomes of model organisms. In silico analyses indicate that <5% of all predicted human proteins do not generate tryptic methionyl peptides that are detectable by mass spectrometers. Methionine can be specifically and quantitatively converted to its sulfoxide derivative by mild oxidation using hydrogen peroxide in an acidic buffer [17] (Figure 2). Only the sulfoxide (and not the sulfone) derivative is formed, and neither cysteine nor tryptophan is oxidized. Peptides with oxidized methionine are more hydrophilic and thus shift during reversed-phase chromatography. When we applied this technique to an Escherichia coli lysate (50 × 106 cells), we identified >800 different proteins [17]: this detection level is an order of magnitude higher than that achieved in analyses of the same amount of starting material by conventional 2D-gel analysis, illustrating the high sensitivity of this non-gel approach. Isolation of peptides containing cysteine To isolate cysteine-containing peptides, the disulfide bridges in proteins are reduced and the free cysteines are reacted with Ellman’s reagent, 5,5′-dithiobis(2-nitrobenzoic acid) [22], before www.drugdiscoverytoday.com

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Isolation of N-terminal peptides Although isolating methionyl or cysteinyl peptides can reduce a sample’s complexity by a factor of 5, this reduction in complexity is not sufficient for cell lysates from higher organisms. As a result, many isolated peptides escape analysis owing to the high peptide flux towards the mass spectrometer. We have therefore adapted the COFRADIC sorting chemistry such that N-terminal peptides are isolated [18]. In this technique, the chemistry applied is slightly more complicated. After protein extraction, free amino groups are blocked by acetylation and thiol groups are blocked by alkylation. Subsequently, the modified proteins are digested with trypsin, which will now cleave only at the carboxylic side of arginine to generate two types of peptide: N-terminal peptides with a blocked α-N-terminus, and internal peptides with a free α-N-terminus (Figure 4). After the primary fractionation, internal peptides are quantitatively modified by 2,4,6-trinitrobenzenesulfonic acid (TNBS) [26,27], which attaches a very hydrophobic trinitrophenyl group to the free N-termini of these peptides. When this peptide mixture is refractionated, the unmodified N-terminal peptides elute in the same time interval as in the primary run. By contrast, the modified internal peptides move out of this interval and elute at much later times (i.e. they undergo a hydrophobic shift). N-terminal peptide isolation, called ‘N-teromics’, is a typical example of ‘reversed COFRADIC’; that is, the selected peptides do not shift, but the bulk of non-selected peptides are induced to shift. N-teromics needs a quantitative modification reaction to avoid contamination by traces of unreacted major components. So far,TNBS is the only reagent that meets this requirement. Nteromics has been used to analyze human thrombocytes, for which it identified 264 proteins and revealed the processing and degradation of several important proteins, as well as the differential N-terminal formylation of two forms of platelet migratory inhibition factor [18].

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Figure 2. Isolation of methionyl peptides by COFRADIC™. After trypsin digestion, the peptide mixture is fractionated and the methionyl peptides in each primary fraction are oxidized to their sulfoxide derivatives. In the secondary runs, these latter peptides undergo a hydrophilic shift (−δ) as compared with the bulk of peptides, which lack methionine.

trypsin digestion. Ellman’s reagent is known to react quantitatively with free thiols [23,24], introducing a hydrophobic moiety on cysteines (Figure 3). After protein digestion, the peptide mixture is fractionated in a primary run. The hydrophobic thionitrobenzoic acid group is then removed by reduction with the strong reducing agent tris(2-carboxyethyl)phosphine (TCEP) before the secondary run [25]. During the secondary runs, cysteinyl peptides undergo a hydrophilic shift and are isolated from the bulk of non-cysteinyl peptides. This procedure has been applied to the analysis of human plasma from which serum albumin and antibodies have been partially depleted by affinity columns. Using <100 µl of plasma, the cysteine COFRADIC procedure identified 102 different proteins [19]. By comparing known concentrations of the identified plasma proteins, we calculated that COFRADIC can simultaneously identify, from a complex protein mixture, proteins that vary in concentration by at least five orders of magnitude. Primary fraction

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Figure 3. Isolation of cysteinyl peptides by COFRADIC™. First, reduced cysteines in the intact proteins are reacted with Ellman’s reagent and then the proteins are digested. All cysteinyl peptides that are present in the primary fractions carry a hydrophobic group that is removed by reduction before the secondary separation. Similar to the isolation of methionyl peptides, cysteinyl peptides undergo a hydrophilic shift (−δ) and are thus specifically collected.

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Isolation of phosphorylated peptides We observed that there is a strong difference in retention behavior on a reversed-phase column between phosphorylated peptides and their unphosphorylated counterparts. Unphosphorylated peptides are more hydrophobic, and this difference forms the basis for the COFRADIC procedure for isolating ex-phosphorylated peptides. After an initial enrichment for phosphorylated peptides, the peptide mixture is fractionated on a reversed-phase column. Primary fractions contain in vivo unphosphorylated peptides (owing to the inefficient first enrichment procedure) in addition to

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Second, sorting of representative peptides with COFRADIC relies on the physicochemical differences between altered and (i) Ac AA1 AA2 AA3 AA4 ... Arg Ac AA1 AA2 AA3 AA4 ... Arg unaltered compounds. These differences Ac AA1 Lys AA3 AA4 ... Arg Ac AA1 Lys AA3 AA4 ... Arg (ii) are exploited in conventional chromatogε NH-Ac ε NH-Ac raphy setups that are easy to control and TNBS well understood; altered peptides elute in NO2 predetermined intervals that can be subdiH2N AA1 AA2 AA3 AA4 ... Arg (iii) NO2 vided into fractions, thereby lowering the NH AA1 AA2 AA3 AA4 ... Arg overall sample complexity. By contrast, NO2 affinity-based peptide isolation uses a chemNO2 ical or biologically active tag to retain a set H2N AA1 AA2 Lys AA4 ... Arg of peptides; in many cases, the isolated NH AA1 AA2 Lys AA4 ... Arg (iv) NO2 ε NH-Ac mixture is still complex and needs further ε NH-Ac NO2 separation by multidimensional chromatoDrug Discovery Today: TARGETS graphic systems [29]. Third, in ICAT-type methodologies, difFigure 4. Specific isolation of N-terminal peptides by COFRADIC. In the primary run, a ferent stable isotopes can be directly incorpeptide mixture generated from the digestion of alkylated and acetylated proteins is fractionated. Each primary fraction contains two types of peptide: N-terminal peptides (i, ii), porated into the tag and thus transferred to and internal peptides (iii, iv). The internal peptides react quantitatively with TNBS, which adds the specified peptide. This provides some a hydrophobic trinitrophenyl group to the α-amino group. In the secondary runs, the modified levels of freedom with respect to both the internal peptides undergo a large hydrophobic shift (+δ) and are thus separated form the unaffected N-terminal peptides, which elute in the same time frame as in the primary run. nature of isotopes used and the number incorporated. In COFRADIC, a more general method is used for differential non-gel phosphorylated ones. After dephosphorylation with a broad proteomics, namely, the trypsin-catalyzed incorporation of heavy range phosphatase such as calf intestinal alkaline phosphatase, isotopes [30].This method leads to the incorporation of two 18O the latter can be specifically shifted to later elution times durisotopes in the carboxyl group of terminal lysine or arginine ing the secondary run and collected for analysis [28]. residues, resulting in a mass difference of 4 Da, which is suffiWe have used this technology to analyze the phosphoprocient to distinguish light and heavy peptide variants and to teome of HepG2 cells and have so far identified 142 phosphomeasure their ratio. peptides belonging to >100 different proteins (K. Gevaert and J. Vandekerckhove, unpublished). Although information about What are the strengths and weaknesses of protein phosphorylation is scarce in protein sequence databases, COFRADIC™? searches of scientific literature indicate that many of the proAlthough COFRADIC uses generally available MS technology teins identified are known phosphoproteins, although their exact to analyze sorted peptides and software tools to identify and phosphorylation sites are unknown. classify proteins, it is particular in the way that samples are prepared. One of its strong points is its flexible approach to pepWhat distinguishes COFRADIC™ from other tide sorting: because it is not linked to tagging chemistry, there technologies? is considerable freedom in selecting specific and quantitative As a peptide sorting device, COFRADIC shares some similarmodification reactions. ities with isotope-coded affinity tag (ICAT) methods [8]: it Because COFRADIC relies on chromatographic methodolselects a subset of peptides as ‘signatures’ for the proteome ogies, we can exploit numerous techniques that have been composition, thereby simplifying the sample mixture; sorting recently developed in this area, such as monolithic columns to is also based on a specific modification of a functional group allow faster separations and completely automated systems for that is present only in a subset of peptides. At the technical sample injection and collecting fractions. Another strength is that level, the COFRADIC and ICAT approaches have essential COFRADIC is unbiased with respect to false positives and to indifferences. complete coverage of the proteome; these factors can be probFirst, the modification introduced in COFRADIC is not lematic for affinity-based methods when, respectively, too harsh necessarily an affinity tag: any specific modification can be and too soft elution conditions are used. In general, COFRADIC used. This versatility provides a very high degree of freedom has proved to be highly sensitive, mainly because it requires very for the selection procedure, as exemplified above. simple steps during which accumulating losses are limited. Primary fraction

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For differential analysis, COFRADIC uses an 18O-incorporation procedure.Trypsin-catalyzed 18O incorporation has been known for a long time [31–33], but only recently has it gained appreciation and been improved for differential non-gel proteomics [30]. This step has the benefit of being extremely simple: it can be carried out on tryptic peptides and does not need special chemistry or the removal of excess of reagents. Two points of concern remain: the first is related to the nongel peptide-based approach; the second is a consequence of the chromatographic fractionation procedure. First, although COFRADIC generates simplified peptide mixtures, these can still be complex and thus can generate a high flux of peptides towards the mass spectrometer. This high flux can lead to incomplete analysis of the proteome, because several peptides can be overlooked.This problem is similarly encountered when affinity-purified peptides are analyzed by MS, and thus it is not intrinsic to COFRADIC. Second, because COFRADIC uses fractionation technology, it is important to ensure that there is a minimal amount of post-column mixing. Indeed, any peak broadening in the first separation will be transferred to and amplified in the second separation, leading to an unwanted diffusion of peptides over several final fractions. Such diffusion unnecessarily complicates the subsequent analysis and reduces the overall sensitivity. It is therefore mandatory to minimize post-column mixing. In our hands, peptides are generally recovered in one or sometimes two fractions after the first run. After modification, the altered peptides can thus appear in two separate intervals; this could slightly reduce the sensitivity but it is not crucial. To cover as much of an isolated proteome as possible in an unambiguous manner, we have built our own suite of bioinformatic tools to improve all aspects of peptide-based protein identification, including database searching by programs such as Mascot [34] in different sequence databases, as well as to improve data repository and the classification of identified proteins according to their biological function or their localization within the cell (developed on the basis of the Gene Ontology Annotation database [35]). More information can be found at http://www.proteomics.be and http://penyfan.ugent.be, and links herein. Who are the main competitors in the field? The proteomics field is extremely vast, and the problems to be solved are both complex and highly diverse. The position is best illustrated by considering humans: the human genome is reported to contain about 30 000 different genes; however, taking into account alternative splicing, protein processing, controlled degradation and posttranslational modifications, the number of different human polypeptides could easily be several millions. Our need to identify these different products, to understand their functions and to distinguish functional from S20

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nonfunctional variants is why proteomics is becoming so important. With the aid of genomic information, we now have a solid basis with which to tackle such problems. In this highly complex field, every verified technology that is newly introduced could easily find its field of application (or ‘niche’), and the compilation and integration of data obtained with different techniques will broaden our insight into the functional proteome. In this context, it is difficult to discuss direct competition: in the extreme case, we can describe only overlapping fields. We estimate that with improvements in zoom gels [36], novel quantitative and differential staining methods [37] and advances in software to analyze protein spot patterns, 2D-PAGE will keep its foot in the door. One of the main points of criticism concerning 2D-PAGE – that it covers only a small part of the proteome – might be true to some extent. But 2D gels have been shown to be reproducible. In many cases, it might be better to compare a small part of the proteome in a reproducible manner than to compare a larger number of proteins with irreproducible coverage. The latter is sometimes observed when a large flux of peptides is sent to a mass spectrometer, as happens in strategies that couple multidimensional liquid chromatography with MS/MS. Such techniques are therefore strong on a qualitative level but still have to demonstrate their robustness for differential proteomics. Preselecting a subset of peptides is clearly a better solution for whole-proteome analysis, provided that the selected peptides cover most (if not all) of the proteins. Through the reduction in sample complexity, a broader coverage and thus higher reproducibility is reached. At this level, COFRADIC and ICAT-based methodologies might be comparable. The ICAT technology depends on specific reagents that are not easily synthesized, although some can be purchased commercially (i.e. Applied Biosystems; http://www.appliedbiosystems.com/). Since the first paper on ICAT was published [8], several laboratories have used this methodology. COFRADIC is more recent [17], and follow-up papers have so far been published by the inventors [18,19]. How can COFRADIC™ be improved? The basic concept of COFRADIC will probably not change much in the future. There is room, however, to improve the sorting chemistries and peptide identification strategies and to create COFRADIC protocols for analyzing various posttranslational modifications and identifying drug targets. To improve the chemistry, we can try novel reaction protocols that have so far not been included in the optimization experiments. For example, in the N-teromics approach (see above and Figure 4), the acetylation step could be replaced by another amine-blocking reaction such as carbamylation. Many modification reactions look promising, but we await determination of

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their specificity and quantitative nature before introducing them into the existing protocols. Recent developments in MS that have improved both accuracy and throughput will add value to COFRADIC. In this respect, we are optimistic about the development of Fourier transform ion cyclotron mass analyzers, which promise a large intraspectrum dynamic range, and unsurpassed sensitivity and accuracy [38]. Another point that needs our attention is the identification of a protein from a complex mixture on the basis of linking a single MS/MS spectrum to one peptide sequence.This problem is not specific to COFRADIC, but is inherent in any peptidebased non-gel proteomic technology. It stresses the need for peptide identification software algorithms such as PINXIT (http://penyfan.ugent.be), which we developed to clean up the results of database searches by holding back only those peptides or proteins that have very high confidence scores. We are currently implementing sorting protocols for N- and O-linked glycopeptides, S-nitrosylated, methylated, S-glutathionylated and ubiquitinated peptides. All of these derivatives are registered as important posttranslational modifications that reflect the biological status of the cell and control many crucial protein functions. Last but not least, COFRADIC can be used to search compounds that can be specifically and covalently linked to their target proteins, paving the way for a vast array of future protocols for screening potential drug targets.

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5 Washburn, M.P. et al. (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242–247 6 Lipton, M.S. et al. (2002) Global analysis of the Deinococcus radiodurans proteome by using accurate mass tags. Proc. Natl.Acad. Sci. U. S.A. 99, 11049–11054 7 Wu, C.C. et al. (2003) A method for the comprehensive proteomic analysis of membrane proteins. Nat. Biotechnol. 21, 532–538 8 Gygi, S.P. et al. (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–999 9 Geng, M. et al. (2000) Signature-peptide approach to detecting proteins in complex mixtures. J. Chromatogr.A. 870, 295–313 10 Ji, J. et al. (2000) Strategy for qualitative and quantitative analysis in proteomics based on signature peptides. J. Chromatogr. B 745, 197–210 11 Spahr, C.S. et al. (2000) Simplification of complex peptide mixtures for proteomic analysis: reversible biotinylation of cysteinyl peptides. Electrophoresis 21, 1635–1650 12 Oda,Y. et al. (2001) Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nat. Biotechnol. 19, 379–382 13 Wang, S. and Regnier, F.E. (2001) Proteomics based on selecting and quantifying cysteine containing peptides by covalent chromatography. J. Chromatogr.A. 924, 345–357 14 Zhou, H. et al. (2001) A systematic approach to the analysis of protein phosphorylation. Nat. Biotechnol. 19, 375–378 15 Ficarro, S.B. et al. (2002) Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 20, 301–305 16 Zhang, H. et al. (2003) Identification and quantification of N-linked

Partnerships in COFRADIC™? The COFRADIC patent was made publicly available on 3 October 2002 [39]. This patent is owned by the Flanders Interuniversity Institute of Biotechnology (http://www.vib.be), which is pursuing industrial applications in terms of both the technology and the products derived from its application. The laboratory itself, although following its own research interests using COFRADIC, remains accessible for academic collaborations.

glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat. Biotechnol. 21, 660–666 17 Gevaert, K. et al. (2002) Chromatographic isolation of methioninecontaining peptides for gel-free proteome analysis: identification of more than 800 Escherichia coli proteins. Mol. Cell. Proteomics 1, 896–903 18 Gevaert, K. et al. (2003) Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides. Nat. Biotechnol. 21, 566–569 19 Gevaert, K. et al. Reversible labeling of cysteine-containing peptides allows their chromatographic isolation for non-gel proteome studies.

Acknowledgements K.G. is a Postdoctoral Fellow of the Fund for Scientific Research – Flanders (Belgium) (F.W.O. – Vlaanderen).

Proteomics (in press) 20 Cruickshank, W.H. et al. (1974) Diagonal chromatography for the selective purification of tyrosyl peptides. Can. J. Biochem. 52, 1013–1017 21 Brown, J.R. and Hartley, B.S. (1966) Location of disulphide bridges by

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