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Peptidomics technologies for human body fluids
TRENDS in Biotechnology Vol.19 No.10 (Suppl.) October 2001
A TRENDS Guide to Proteomics
|
Review
Peptidomics technologies for human body fluids Michael Schrader and Peter Schulz-Knappe Peptides play a central role in many physiological processes. In order to analyse comprehensively all peptides and small proteins of a whole organism or a subsystem (peptidome), the use of technologies other than 2D gel electrophoresis is necessary. Approaches that use liquid chromatography or affinity purification and mass spectrometric identification have now been developed and applied successfully to the analysis of human body fluids. Together with the establishment of the human genome as the blueprint for human life, knowledge about the functionally active substances in our body that have evolved from the genome becomes more and more important. It is therefore necessary that one aspect of the human proteome project be directed towards improving our understanding of the molecules that make up our complex regulatory systems and, ultimately, our life. For decades, many research teams have searched for the biochemical messenger molecules that organize the myriad of regulatory processes in our body, such as peptide hormones, neuropeptides, cytokines and enzyme inhibitors.These molecules are necessary for the communication between all the different specialized cell types that make up the subsystems of the whole body – fascinating machinery that no engineer is able to duplicate. The transport of these messengers is most often performed through body fluids that enable communication even between cells that are too remote to interact directly or by migration. There has been significant progression in the field of proteomics since the research of Bayliss and Starling or Banting and Best during the beginning of the last century, and since the sequences of secretin and insulin (two of the first prominent examples for the family of peptide hormones) were determined decades later1. The technologies for isolation and identification that have evolved have led to the discovery of several important classes of peptide hormones, for example, gut hormones like secretin and cholecystokinin, neuropeptides such as thyrotropin releasing hormone and leuteinizing hormone http://trends.com
releasing hormone, and the growth hormone somatostatin1. These few examples demonstrate that peptides are of paramount importance for many physiological processes (Table 1). Since then, much progress has been made; however, we have still to achieve a sound knowledge about human peptides and their functions. One reason is the lack of technologies that enable a comprehensive analysis of peptides.The classical discovery strategies for peptides have been purifications from tissue extracts guided by function, and almost always, many steps are needed (which often take years) to generate a new sequence.This can be attributed to the complexity of biological sources, the small concentration of single components and the overwhelming amounts of a few housekeeping proteins. With the establishment of tools for protein purification (i.e. chromatography and electrophoresis) and protein analysis [i.e. N-terminal chemical Edman sequencing of almost pure proteins and peptides, and mass spectrometry (MS) for identification and characterization], the tools for peptide research have become much more sophisticated. Moreover, the ability to search expressed sequence tag (EST) and genome databases has made the identification much easier. Nevertheless, the subdiscipline of peptidomics is a still growing field of research, although not very well explored. From the estimated 30 000 genes in the human genome, around three times the number of proteins are expected to be identified, owing to variations that occur during transcription and translation2. Further post-translational and proteolytic processing3,4 should lead to the discovery of several hundred thousand to
0167-7799/01/$ – see front matter ©2001 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(01)01800-5
Michael Schrader* and Peter SchulzKnappe BioVisioN GmbH & Co. KG, Feodor-Lynen-Str. 5, D-30625 Hannover, Germany. *e-mail: m.schrader@ biovision.de
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Review
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A TRENDS Guide to Proteomics
TRENDS in Biotechnology Vol.19 No.10 (Suppl.) October 2001
Table 1. Examples of peptides and small proteins in human body fluids and their physiological relevance Peptide
Molecular mass (kDa)
Physiological relevance
Thyrotropin-releasing hormone (TRH)33
0.4
Regulation of thyroid hormones
Oxytocin33
1.0
Contraction of the uterus and stimulation of lactation
Angiotensin II (Ref. 33)
1.0
Blood pressure regulation
Calcitonin33
3.5
Bone turnover
Cholecystokinin33
3.9
Flow of bile and exocrine pancreatic secretion
Insulin33
5.8
Blood glucose regulation
Insulin-like growth factor I (Ref. 33)
7.5
Body growth
Parathyroid hormone33
9.4
Bone turnover
Serum-amyloid protein34
12
Inflammation
millions of different peptides that exist in the human body. A notable number of them should be of potential use diagnostically or therapeutically; amyloid-β peptides are proposed to be diagnostic markers for Alzheimer’s disease5 and the recently discovered peptide hormone resistin6 is assumed to be of relevance to obesity and diabetes. Extending proteomics with technologies for the analysis of peptides Over recent years, 2D gel electrophoresis in combination with MS has become the main proteomics research tool7. This method analyses medium-sized proteins that range from 10 to 200 kDa with an isoelectric point (pI) between 4 and 10. However, smaller proteins and native peptides are not yet covered by standard proteomics methods8. As there is no clear-cut definition of a peptide [from, for example, the International Union of Pure and Applied Chemistry (IUPAC)], the term peptide will be used throughout this article to refer to peptides and small proteins of <1 kDa to molecules of about 20 kDa. Owing to their physico-chemical properties and the shortcomings of gel technologies, only a few faint spots that correspond to peptides of <20 kDa are visible on a gel image. Development of proteomic methods that deal with these smaller molecules has not yet attracted a lot of attention, which does not correlate with the importance of known and expected substances in this range. For example, most of the important biopharmaceutical products approved for therapeutic applications are molecules within a molecular mass range of 1–40 kDa (Ref. 9). Our definition of peptides as small proteins with a molecular mass that is less than ~20 kDa has its implications,
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because the physico-chemical properties of these molecules are different from proteins. For example, the mobility of peptides is higher than that of larger proteins, which makes it very difficult to focus them in gels, and their ability to bind the different stains used in visualization procedures is small. Another important factor is the stability of peptides, which tend not to denature irreversibly. In addition, the folding behaviour of peptides in solution, as well as during adsorption and desorption processes, is mostly reversible. However, their generally lower hydrophobicity enables them to dissolve easily in aqueous systems without the use of detergents. Therefore, the most prominent separation technology for molecules of up to about 20 kDa is liquid chromatography (LC), in particular, reversed-phase and ion-exchange chromatography8,10. Another aspect of the lower hydrophobicity of smaller peptides is that modifications to improve solubility, such as glycosylation and phosphorylation, are less common than in proteins. Alternatively, disulfide bonds, which determine a specific spatial structure, are often found (for example, in peptide hormones, chemokines or defensins). Other modifications, such as N-terminal pyroglutamylation or C-terminal amidation, are introduced to protect these molecules from proteolysis by amino- or carboxypeptidases during their transport from the site of release to the site of action. Consequently, different analytical strategies have to be applied to peptides when compared with larger proteins. After successful extraction and purification of peptides, the next important aspect is sequence identification. Presently, most sequence information is generated by MS followed by database comparison, http://trends.com
TRENDS in Biotechnology Vol.19 No.10 (Suppl.) October 2001
owing to the high-throughput nature of the technique (when compared with Edman sequencing or amino acid analysis11). Proteins are too large to be analysed directly by MS, and hence, are usually converted into several peptides by specific proteolytic enzymes, and identified by comparison of the generated characteristic cleavage pattern within databases. For peptides, the number of possible specific internal cleavages is typically too small for significant identification using databases; therefore, peptides are identified by their characteristic fragment pattern after collision-induced dissociation in MS–MS experiments12. To circumvent the shortcomings of protein identification after separation by 2D gel electrophoresis, some effort has been made to apply LC to enzymatically-treated samples, in order to convert proteins to peptides. As shown successfully for yeast13, the whole protein content of a source can be trypsinated and submitted to multidimensional chromatography with subsequent MS–MS analysis and identification. With the improvements of instrument performance and software for database searches14, peptides with molecular masses of up to 10 kDa have already become accessible. Although ion-trap instruments are widely used in LC–MS–MS and direct MS–MS, identification of peptides as enzymatic fragments of proteins, limited resolution of higher charge states, restricted fragment ion resolution and mass accuracy, make these instruments second choice for analysis of (native) peptides that are greater than 3–4 kDa. Hybrid quadrupole TOF mass spectrometers are preferentially used because of their high mass accuracy and resolution11,15. An alternative approach is the use of Fourier-transform ion cyclotron resonance MS (Ref. 16), although this still lacks an adequate throughput because of limited automization capability. The existing databases, whether they cover proteins, genomes or ESTs, are of great help in the acceleration and automation of identification processes, although there is still a lack of specific databases and software for searching through for more peptide-specific information. At present, post-translational modifications (PTMs), proteolytic processing and amino acid variations, are recorded only as annotations in databases and thus cannot be searched systematically.This prevents automatic identification of many peptides, even though a good and representative MS–MS spectrum might be available. This issue is especially important for peptides and needs to be addressed in the future. Extracellular fluids in humans Physiological and pathological changes are reflected in the production and the metabolism of proteins and peptides. Such changes are detectable in extracellular fluids, http://trends.com
A TRENDS Guide to Proteomics
|
Review
which represent the major link between all cells, tissues and organs of the human body3. Analysis by genomic or transcriptomic methods is of little use, because body fluids are a collection of substances from a variety of cell types that arise as a consequence of transport or diffusion and further processing. The development of proteomic technologies to analyse the dynamics of these systems is of significant interest, because these technologies represent the multitude of concerted biological processes in a living organism7,8. For the measurement of proteins within the range of 10–200 kDa, protocols using 2D gel electrophoresis are well established7. Recently, a technology became available for the comprehensive analysis of peptides between 0.5 and 20 kDa in human body fluids, which play a pivotal role in many physiological processes8. By identifying specific changes in the peptide and protein composition of human body fluids using quantitative differential display methods, it has now become feasible to discover novel biomarkers that indicate diseases and other conditions, and to identify drug candidates and applications for drug development. Peptides in blood plasma Blood is the major link between all cells, tissues and organs of humans and contains the most representative collection of peptides, proteins and protein fragments that are produced in the entire body. Release of peptidic or protein compounds into the extracellular space is one of the most important mechanisms by which communication between cells and organs is established and maintained. Knowledge about the housekeeping plasma proteins, such as albumin, fibrinogen, immunoglobulins and others that are present in large concentrations, is well established. However, the identification of peptides and proteins with regulatory function is difficult because they occur at low concentrations and within a complex mixture of the main plasma proteins. Different strategies have been applied to overcome this issue. These include isolations that are directed by screening for a specific biological activity, immunological detection, binding to orphan receptors or chemical specificity, and have led to the discovery of many important peptide hormones, cytokines and growth factors8. With the emergence of the Human Genome Project, a comprehensive approach to unravelling this biological source has become an issue of high medical importance. To this end, a basis for the systematic purification of all peptides in blood plasma has been established using haemofiltrate as a suitable source3. This blood filtrate (with a cut-off at about 20–30 kDa) is generated from individuals with end-stage renal disease and then
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A TRENDS Guide to Proteomics
TRENDS in Biotechnology Vol.19 No.10 (Suppl.) October 2001
analysed with MS methods17. Multi-dimensional maps of more than 5000 distinct components were compiled using matrix-assisted laser desorption–ionization (MALDI)–time-of-flight (TOF)–MS or on-line LC–MS (Ref. 18), and subsequent identification by tandem MS experiments has resulted in a database of human blood peptides. Of 340 sequence entries registered in a first publication from 1999 (Ref. 19), 55% were fragments of known plasma proteins, but a substantial part (12%) were members of peptide families such as cytokines, defensins or peptide hormones – some of them as-yet unknown compounds. Interestingly, several of the discovered new molecules occur at nanomolar concentrations and are thus very abundant.
Clinical sample
Peptide extraction and separation of proteins
HPLC separation
MALDI–MS
96 mass spectra per sample Transformation of data
Peptide mass fingerprint Differential display of multiple samples Similarities and differences TRENDS in Biotechnology
Figure 1. Schematic representation of the underlying process for a differential display of complex peptide mixtures Peptides are extracted from a clinical sample and separated into 96 fractions using reversed-phase chromatography. All fractions are measured by matrix-assisted laser desorption–ionization (MALDI)–time-of-flight (TOF)–mass spectrometry (MS) and data are transformed into a 2D gel-like picture by means of a specialized software. Typically, 1000–5000 components in the range of 0.5–15.0 kDa are visualized and defined by the three dimensions: (1) chromatographic fraction number; (2) mass per charge ratio; and (3) intensity from MS data. Differential comparison of the resulting peptide mass fingerprint is achieved by comparing the components detected by calculating signal heights and/or areas, using appropriate internal and external calibration and standardization procedures.
purified. The plasma protein content is reduced by a factor of 1000, but regulatory compounds remain present at their normal physiological concentrations. Moreover, haemofiltrate contains little to no enzymatic activity, and is quickly acidified and cooled to represent a ‘frozen’ status of blood peptides. The availability of haemofiltrate in large amounts has made possible the establishment of a ‘peptide bank’ comprising a collection of blood peptides in pre-fractionated batches from several thousand litres of blood filtrate processed in a reproducible manner. This material was systematically
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Other body fluids Several other human body fluids are also rich in peptides. Of particular interest for diagnostic purposes are easily accessible sources, such as urine, lacrimal fluid or saliva. For example, several biochemical markers in urine are used to measure bone turnover20. Another example is the successful application of surfaceenhanced laser desorption–ionization (SELDI) technology for the identification of a defensin peptide of 3.4 kDa in urine as a biomarker for transitional cell carcinoma of the bladder21. In a systematic approach, peptides in urine have been characterized and identified by using LC–MS and Edman sequencing22. Recently, a LC–MS–MS strategy has been used towards defining a urinary proteome using unfractionated tryptic digests 23. Specific peptides in saliva have been linked to diseases of the teeth (e.g. caries)24, and human breast milk is a rich source of many peptide growth hormones and antibiotics25. Less accessible liquid subsystems in the body, such as cerebrospinal or synovial fluid, are the first sources to be screened in order to understand local physiological processes and diseases. Cerebrospinal fluid is rich in neuropeptides and, with its relevance to neurodegenerative diseases5, small amounts (less than 1 ml) have already been analysed. Comprehensive methods have been used to show a specific peptide pattern8 and many peptide components have been generated by specific proteolytic processing26. For synovial fluid, a few characteristic peptide biomarkers are already known20 but a comprehensive peptidomic analysis is still lacking. Quantitative approaches Quantitative analysis of selected components A very promising feature of peptidomic analysis is that several methods for quantitative analysis are already http://trends.com
TRENDS in Biotechnology Vol.19 No.10 (Suppl.) October 2001
available. Besides the immunoassay-based measurement with radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), MS can now be used to determine the amounts of specific peptides with the modern instruments that are capable of both improving the purification and performing the quantification. The initial purification can be performed by affinity27,28 or chromatographic24 purification, with the analysis performed either on- or off-line. It has been shown that the quantitative measurement can be based not only on electrospray MS (Ref. 29), but also on MALDI–TOF–MS (Refs 27,28) detection methods. The quantification is performed by comparison with external27 or internal28,29 references. A less specific method using diodearray detection during reversed-phase chromatography has been described for the quantification of several salivary peptides30.
A TRENDS Guide to Proteomics
|
Review
be possible to distinguish between different types or individual variations of a disease, such as tumours and their ability to undergo metastases. Finally, peptidomes from body fluids will contribute to the human proteome initiative, and will thus become an important tool in pharmaceutical development. References 1 Crapo, L. (1985) Hormones: The Messengers of Life, W.H. Freeman 2 Galas, D.J. (2001) Making sense of the sequence. Science 291, 1257–1260 3 Schulz-Knappe, P. et al. (1996) Systematic isolation of circulating human peptides: the concept of Peptide Trapping. Eur. J. Med. Res. 1, 223–236 4 Hook, V.Y., ed. (1998) Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing, R.G. Landes 5 Knopman, D. (2001) Cerebrospinal fluid beta-amyloid and tau proteins for the diagnosis of Alzheimer disease. Arch. Neurol.
Differential display of complex mixtures A few years ago, two initial publications appeared that clearly demonstrated the capability of MALDI–TOF–MS in semiquantitative analysis of complex peptide mixtures31,32. Although a differential display had been shown only for a few different spectra of rat neurointermediate lobes31 and the haemolymph of Drosophila32, it became clear that an application for other biological sources, such as body fluids, could become possible. With the development of a robust and reproducible separation technology for peptides in complex biological sources by adsorption or affinity purification on a chip21, chromatography yielding a 3D display8 (Fig. 1) and more sophisticated data-mining tools, the comprehensive discovery of human peptides relevant as biomarkers or drug targets has just begun.
58, 373–379 6 Steppan, C.M. et al. (2001) The hormone resistin links obesity to diabetes. Nature 409, 307–312 7 Kennedy, S. (2001) Proteomic profiling from human samples: the body fluid alternative. Toxicol. Lett. 120, 379–384 8 Schulz-Knappe, P. et al. (2001) Peptidomics: the comprehensive analysis of peptides in complex biological mixtures. Comb. Chem. High Throughput Screen. 4, 207–217 9 Walsh, G. (1998) Biopharmaceuticals: Biochemistry and Biotechnology, John Wiley & Sons 10 Lundell, N. (1995) A peptide in a haystack – strategies for a biochromatographer. LC GC 8, 636–647 11 Blackstock, W.P. (2000) Trends in automation and mass spectrometry for proteomics. In Proteomics: A Trends Guide (Blackstock, W. and Mann, M., eds), pp. 12–17, Elsevier 12 Papayannopoulos, I.A. (1995) The interpretation of collisioninduced dissociation tandem mass spectra of peptides. Mass Spectrom.
Prospects The application of peptidomic approaches to analyse human body fluids will complement efforts to study gene expression using proteomic or transcriptomic technologies. It will serve as a tool to study gene expression products in a relevant and complex class of clinically interesting molecules, with the potential for application in clinical analysis and drug development. The knowledge gained from analysis of the peptide content in human body fluids will have a direct impact on the diagnosis of many diseases. In the future, the old paradigm of one disease related to a single gene and diagnosed by a single marker or treated by a single drug, will no longer be valid. The identification of interacting gene expression networks and the measurement of marker patterns must be achieved in order to differentiate between diseases. Using such tools, it will http://trends.com
Rev. 14, 49–73 13 Washburn, M.P. et al. (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242–247 14 Beavis, R.C. and Fenyö, D. (2000) Database searching with massspectrometric information. In Proteomics: A Trends Guide (Blackstock, W. and Mann, M., eds), pp. 22–27, Elsevier 15 Verhaert, P. et al. (2001) Matrix-assisted laser desorption/ionization quadrupole time-of-flight mass spectrometry: an elegant tool for peptidomics. Proteomics 1, 118–131 16 Conrads, T.P. et al. (2000) Utility of accurate mass tags for proteome-wide protein identification. Anal. Chem. 72, 3349–3354 17 Jürgens, M. et al. (1997) Multi-dimensional mapping of human blood peptides by mass spectrometry. J. Biomol. Tech. 9, 24–30 18 Raida, M. et al. (1999) Liquid chromatography and electrospray mass spectrometric mapping of peptides from human plasma filtrate Am. Soc. Mass Spectrom. 10, 45–54
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19 Richter, R. et al. (1999) Composition of the peptide fraction in human blood plasma: data base of circulating human peptides. J. Chromatogr. B 726, 25–35 20 Garnero, P. et al. (2000) Molecular basis and clinical use of
β-2-microglobulin using mass spectrometric immunoassay. Anal. Biochem. 289, 26–35 28 Gobom, J. et al. (2000) Detection and quantification of
biochemical markers of bone, cartilage, and synovium in joint
neurotensin in human brain tissue by MALDI–TOF–MS. Anal. Chem.
diseases. Arthritis Rheum. 43, 953–968
72, 3320–3326
21 Vlahou, A. et al. (2001) Development of a novel proteomic approach
29 Fierens, C. et al. (2000) Quantitative analysis of urinary C-peptide by
for the detection of transitional cell carcinoma of the bladder in
liquid chromatography-tandem mass spectrometry with a stable
urine. Am. J. Pathol. 158, 1491–1502 22 Heine, G. et al. (1997) Mapping of peptides and protein fragments in
isotopically labelled internal standard. J. Chromatogr. A 896, 275–278 30 Castagnola, M. et al. (2001) Determination of the human salivary
human urine using liquid chromatography-mass spectrometry.
peptides histatin 1, 3, 5 and statherin by high-performance liquid
J. Chromatogr. A 776, 117–124
chromatography and by diode-array detection. J. Chromatogr. B
23 Spahr, C.S. et al. (2001) Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry I. Profiling an unfractionated tryptic digest. Proteomics 1, 93–107 24 Ayad, M. et al. (2000) The association of basic proline-rich peptides from human parotid gland secretions with caries experience. J. Dent. Res. 79, 976–982 25 Britton, J.R. and Kastin, A.J. (1991) Biologically active polypeptides in milk. Am. J. Med. Sci. 301, 124–132 26 Stark, M. et al. (2001) Peptide repertoire of human cerebrospinal fluid: novel proteolytic fragments of neuroendocrine proteins. J. Chromatogr. B 754, 357–367
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27 Tubbs, K.A. et al. (2001) Detection and quantification of
751, 153–160 31 Jimenez, C.R. et al. (1997) Pattern changes of pituitary peptides in rat after salt-loading as detected by means of direct, semiquantitative mass spectrometric profiling. Proc. Natl. Acad. Sci. U. S. A. 94, 9481–9486 32 Uttenweiler-Joseph, S. et al. (1998) Differential display of peptides induced during the immune response of Drosophila: a matrix-assisted laser desorption ionization time-of-flight mass spectrometry study. Proc. Natl. Acad. Sci. U. S. A. 95, 11342–11347 33 König, W. (1993) Peptide and Protein Hormones, VCH Verlagsgesellschaft 34 Cunnane, G. (2001) Amyloid precursors and amyloidosis in inflammatory arthritis. Curr. Opin. Rheumatol. 13, 67–73
http://trends.com
A TRENDS Guide to Proteomics
|
Review
Peptidomics technologies for human body fluids Michael Schrader and Peter Schulz-Knappe Peptides play a central role in many physiological processes. In order to analyse comprehensively all peptides and small proteins of a whole organism or a subsystem (peptidome), the use of technologies other than 2D gel electrophoresis is necessary. Approaches that use liquid chromatography or affinity purification and mass spectrometric identification have now been developed and applied successfully to the analysis of human body fluids. Together with the establishment of the human genome as the blueprint for human life, knowledge about the functionally active substances in our body that have evolved from the genome becomes more and more important. It is therefore necessary that one aspect of the human proteome project be directed towards improving our understanding of the molecules that make up our complex regulatory systems and, ultimately, our life. For decades, many research teams have searched for the biochemical messenger molecules that organize the myriad of regulatory processes in our body, such as peptide hormones, neuropeptides, cytokines and enzyme inhibitors.These molecules are necessary for the communication between all the different specialized cell types that make up the subsystems of the whole body – fascinating machinery that no engineer is able to duplicate. The transport of these messengers is most often performed through body fluids that enable communication even between cells that are too remote to interact directly or by migration. There has been significant progression in the field of proteomics since the research of Bayliss and Starling or Banting and Best during the beginning of the last century, and since the sequences of secretin and insulin (two of the first prominent examples for the family of peptide hormones) were determined decades later1. The technologies for isolation and identification that have evolved have led to the discovery of several important classes of peptide hormones, for example, gut hormones like secretin and cholecystokinin, neuropeptides such as thyrotropin releasing hormone and leuteinizing hormone http://trends.com
releasing hormone, and the growth hormone somatostatin1. These few examples demonstrate that peptides are of paramount importance for many physiological processes (Table 1). Since then, much progress has been made; however, we have still to achieve a sound knowledge about human peptides and their functions. One reason is the lack of technologies that enable a comprehensive analysis of peptides.The classical discovery strategies for peptides have been purifications from tissue extracts guided by function, and almost always, many steps are needed (which often take years) to generate a new sequence.This can be attributed to the complexity of biological sources, the small concentration of single components and the overwhelming amounts of a few housekeeping proteins. With the establishment of tools for protein purification (i.e. chromatography and electrophoresis) and protein analysis [i.e. N-terminal chemical Edman sequencing of almost pure proteins and peptides, and mass spectrometry (MS) for identification and characterization], the tools for peptide research have become much more sophisticated. Moreover, the ability to search expressed sequence tag (EST) and genome databases has made the identification much easier. Nevertheless, the subdiscipline of peptidomics is a still growing field of research, although not very well explored. From the estimated 30 000 genes in the human genome, around three times the number of proteins are expected to be identified, owing to variations that occur during transcription and translation2. Further post-translational and proteolytic processing3,4 should lead to the discovery of several hundred thousand to
0167-7799/01/$ – see front matter ©2001 Elsevier Science Ltd. All rights reserved. PII: S0167-7799(01)01800-5
Michael Schrader* and Peter SchulzKnappe BioVisioN GmbH & Co. KG, Feodor-Lynen-Str. 5, D-30625 Hannover, Germany. *e-mail: m.schrader@ biovision.de
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Review
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A TRENDS Guide to Proteomics
TRENDS in Biotechnology Vol.19 No.10 (Suppl.) October 2001
Table 1. Examples of peptides and small proteins in human body fluids and their physiological relevance Peptide
Molecular mass (kDa)
Physiological relevance
Thyrotropin-releasing hormone (TRH)33
0.4
Regulation of thyroid hormones
Oxytocin33
1.0
Contraction of the uterus and stimulation of lactation
Angiotensin II (Ref. 33)
1.0
Blood pressure regulation
Calcitonin33
3.5
Bone turnover
Cholecystokinin33
3.9
Flow of bile and exocrine pancreatic secretion
Insulin33
5.8
Blood glucose regulation
Insulin-like growth factor I (Ref. 33)
7.5
Body growth
Parathyroid hormone33
9.4
Bone turnover
Serum-amyloid protein34
12
Inflammation
millions of different peptides that exist in the human body. A notable number of them should be of potential use diagnostically or therapeutically; amyloid-β peptides are proposed to be diagnostic markers for Alzheimer’s disease5 and the recently discovered peptide hormone resistin6 is assumed to be of relevance to obesity and diabetes. Extending proteomics with technologies for the analysis of peptides Over recent years, 2D gel electrophoresis in combination with MS has become the main proteomics research tool7. This method analyses medium-sized proteins that range from 10 to 200 kDa with an isoelectric point (pI) between 4 and 10. However, smaller proteins and native peptides are not yet covered by standard proteomics methods8. As there is no clear-cut definition of a peptide [from, for example, the International Union of Pure and Applied Chemistry (IUPAC)], the term peptide will be used throughout this article to refer to peptides and small proteins of <1 kDa to molecules of about 20 kDa. Owing to their physico-chemical properties and the shortcomings of gel technologies, only a few faint spots that correspond to peptides of <20 kDa are visible on a gel image. Development of proteomic methods that deal with these smaller molecules has not yet attracted a lot of attention, which does not correlate with the importance of known and expected substances in this range. For example, most of the important biopharmaceutical products approved for therapeutic applications are molecules within a molecular mass range of 1–40 kDa (Ref. 9). Our definition of peptides as small proteins with a molecular mass that is less than ~20 kDa has its implications,
S56
because the physico-chemical properties of these molecules are different from proteins. For example, the mobility of peptides is higher than that of larger proteins, which makes it very difficult to focus them in gels, and their ability to bind the different stains used in visualization procedures is small. Another important factor is the stability of peptides, which tend not to denature irreversibly. In addition, the folding behaviour of peptides in solution, as well as during adsorption and desorption processes, is mostly reversible. However, their generally lower hydrophobicity enables them to dissolve easily in aqueous systems without the use of detergents. Therefore, the most prominent separation technology for molecules of up to about 20 kDa is liquid chromatography (LC), in particular, reversed-phase and ion-exchange chromatography8,10. Another aspect of the lower hydrophobicity of smaller peptides is that modifications to improve solubility, such as glycosylation and phosphorylation, are less common than in proteins. Alternatively, disulfide bonds, which determine a specific spatial structure, are often found (for example, in peptide hormones, chemokines or defensins). Other modifications, such as N-terminal pyroglutamylation or C-terminal amidation, are introduced to protect these molecules from proteolysis by amino- or carboxypeptidases during their transport from the site of release to the site of action. Consequently, different analytical strategies have to be applied to peptides when compared with larger proteins. After successful extraction and purification of peptides, the next important aspect is sequence identification. Presently, most sequence information is generated by MS followed by database comparison, http://trends.com
TRENDS in Biotechnology Vol.19 No.10 (Suppl.) October 2001
owing to the high-throughput nature of the technique (when compared with Edman sequencing or amino acid analysis11). Proteins are too large to be analysed directly by MS, and hence, are usually converted into several peptides by specific proteolytic enzymes, and identified by comparison of the generated characteristic cleavage pattern within databases. For peptides, the number of possible specific internal cleavages is typically too small for significant identification using databases; therefore, peptides are identified by their characteristic fragment pattern after collision-induced dissociation in MS–MS experiments12. To circumvent the shortcomings of protein identification after separation by 2D gel electrophoresis, some effort has been made to apply LC to enzymatically-treated samples, in order to convert proteins to peptides. As shown successfully for yeast13, the whole protein content of a source can be trypsinated and submitted to multidimensional chromatography with subsequent MS–MS analysis and identification. With the improvements of instrument performance and software for database searches14, peptides with molecular masses of up to 10 kDa have already become accessible. Although ion-trap instruments are widely used in LC–MS–MS and direct MS–MS, identification of peptides as enzymatic fragments of proteins, limited resolution of higher charge states, restricted fragment ion resolution and mass accuracy, make these instruments second choice for analysis of (native) peptides that are greater than 3–4 kDa. Hybrid quadrupole TOF mass spectrometers are preferentially used because of their high mass accuracy and resolution11,15. An alternative approach is the use of Fourier-transform ion cyclotron resonance MS (Ref. 16), although this still lacks an adequate throughput because of limited automization capability. The existing databases, whether they cover proteins, genomes or ESTs, are of great help in the acceleration and automation of identification processes, although there is still a lack of specific databases and software for searching through for more peptide-specific information. At present, post-translational modifications (PTMs), proteolytic processing and amino acid variations, are recorded only as annotations in databases and thus cannot be searched systematically.This prevents automatic identification of many peptides, even though a good and representative MS–MS spectrum might be available. This issue is especially important for peptides and needs to be addressed in the future. Extracellular fluids in humans Physiological and pathological changes are reflected in the production and the metabolism of proteins and peptides. Such changes are detectable in extracellular fluids, http://trends.com
A TRENDS Guide to Proteomics
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Review
which represent the major link between all cells, tissues and organs of the human body3. Analysis by genomic or transcriptomic methods is of little use, because body fluids are a collection of substances from a variety of cell types that arise as a consequence of transport or diffusion and further processing. The development of proteomic technologies to analyse the dynamics of these systems is of significant interest, because these technologies represent the multitude of concerted biological processes in a living organism7,8. For the measurement of proteins within the range of 10–200 kDa, protocols using 2D gel electrophoresis are well established7. Recently, a technology became available for the comprehensive analysis of peptides between 0.5 and 20 kDa in human body fluids, which play a pivotal role in many physiological processes8. By identifying specific changes in the peptide and protein composition of human body fluids using quantitative differential display methods, it has now become feasible to discover novel biomarkers that indicate diseases and other conditions, and to identify drug candidates and applications for drug development. Peptides in blood plasma Blood is the major link between all cells, tissues and organs of humans and contains the most representative collection of peptides, proteins and protein fragments that are produced in the entire body. Release of peptidic or protein compounds into the extracellular space is one of the most important mechanisms by which communication between cells and organs is established and maintained. Knowledge about the housekeeping plasma proteins, such as albumin, fibrinogen, immunoglobulins and others that are present in large concentrations, is well established. However, the identification of peptides and proteins with regulatory function is difficult because they occur at low concentrations and within a complex mixture of the main plasma proteins. Different strategies have been applied to overcome this issue. These include isolations that are directed by screening for a specific biological activity, immunological detection, binding to orphan receptors or chemical specificity, and have led to the discovery of many important peptide hormones, cytokines and growth factors8. With the emergence of the Human Genome Project, a comprehensive approach to unravelling this biological source has become an issue of high medical importance. To this end, a basis for the systematic purification of all peptides in blood plasma has been established using haemofiltrate as a suitable source3. This blood filtrate (with a cut-off at about 20–30 kDa) is generated from individuals with end-stage renal disease and then
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analysed with MS methods17. Multi-dimensional maps of more than 5000 distinct components were compiled using matrix-assisted laser desorption–ionization (MALDI)–time-of-flight (TOF)–MS or on-line LC–MS (Ref. 18), and subsequent identification by tandem MS experiments has resulted in a database of human blood peptides. Of 340 sequence entries registered in a first publication from 1999 (Ref. 19), 55% were fragments of known plasma proteins, but a substantial part (12%) were members of peptide families such as cytokines, defensins or peptide hormones – some of them as-yet unknown compounds. Interestingly, several of the discovered new molecules occur at nanomolar concentrations and are thus very abundant.
Clinical sample
Peptide extraction and separation of proteins
HPLC separation
MALDI–MS
96 mass spectra per sample Transformation of data
Peptide mass fingerprint Differential display of multiple samples Similarities and differences TRENDS in Biotechnology
Figure 1. Schematic representation of the underlying process for a differential display of complex peptide mixtures Peptides are extracted from a clinical sample and separated into 96 fractions using reversed-phase chromatography. All fractions are measured by matrix-assisted laser desorption–ionization (MALDI)–time-of-flight (TOF)–mass spectrometry (MS) and data are transformed into a 2D gel-like picture by means of a specialized software. Typically, 1000–5000 components in the range of 0.5–15.0 kDa are visualized and defined by the three dimensions: (1) chromatographic fraction number; (2) mass per charge ratio; and (3) intensity from MS data. Differential comparison of the resulting peptide mass fingerprint is achieved by comparing the components detected by calculating signal heights and/or areas, using appropriate internal and external calibration and standardization procedures.
purified. The plasma protein content is reduced by a factor of 1000, but regulatory compounds remain present at their normal physiological concentrations. Moreover, haemofiltrate contains little to no enzymatic activity, and is quickly acidified and cooled to represent a ‘frozen’ status of blood peptides. The availability of haemofiltrate in large amounts has made possible the establishment of a ‘peptide bank’ comprising a collection of blood peptides in pre-fractionated batches from several thousand litres of blood filtrate processed in a reproducible manner. This material was systematically
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Other body fluids Several other human body fluids are also rich in peptides. Of particular interest for diagnostic purposes are easily accessible sources, such as urine, lacrimal fluid or saliva. For example, several biochemical markers in urine are used to measure bone turnover20. Another example is the successful application of surfaceenhanced laser desorption–ionization (SELDI) technology for the identification of a defensin peptide of 3.4 kDa in urine as a biomarker for transitional cell carcinoma of the bladder21. In a systematic approach, peptides in urine have been characterized and identified by using LC–MS and Edman sequencing22. Recently, a LC–MS–MS strategy has been used towards defining a urinary proteome using unfractionated tryptic digests 23. Specific peptides in saliva have been linked to diseases of the teeth (e.g. caries)24, and human breast milk is a rich source of many peptide growth hormones and antibiotics25. Less accessible liquid subsystems in the body, such as cerebrospinal or synovial fluid, are the first sources to be screened in order to understand local physiological processes and diseases. Cerebrospinal fluid is rich in neuropeptides and, with its relevance to neurodegenerative diseases5, small amounts (less than 1 ml) have already been analysed. Comprehensive methods have been used to show a specific peptide pattern8 and many peptide components have been generated by specific proteolytic processing26. For synovial fluid, a few characteristic peptide biomarkers are already known20 but a comprehensive peptidomic analysis is still lacking. Quantitative approaches Quantitative analysis of selected components A very promising feature of peptidomic analysis is that several methods for quantitative analysis are already http://trends.com
TRENDS in Biotechnology Vol.19 No.10 (Suppl.) October 2001
available. Besides the immunoassay-based measurement with radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), MS can now be used to determine the amounts of specific peptides with the modern instruments that are capable of both improving the purification and performing the quantification. The initial purification can be performed by affinity27,28 or chromatographic24 purification, with the analysis performed either on- or off-line. It has been shown that the quantitative measurement can be based not only on electrospray MS (Ref. 29), but also on MALDI–TOF–MS (Refs 27,28) detection methods. The quantification is performed by comparison with external27 or internal28,29 references. A less specific method using diodearray detection during reversed-phase chromatography has been described for the quantification of several salivary peptides30.
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be possible to distinguish between different types or individual variations of a disease, such as tumours and their ability to undergo metastases. Finally, peptidomes from body fluids will contribute to the human proteome initiative, and will thus become an important tool in pharmaceutical development. References 1 Crapo, L. (1985) Hormones: The Messengers of Life, W.H. Freeman 2 Galas, D.J. (2001) Making sense of the sequence. Science 291, 1257–1260 3 Schulz-Knappe, P. et al. (1996) Systematic isolation of circulating human peptides: the concept of Peptide Trapping. Eur. J. Med. Res. 1, 223–236 4 Hook, V.Y., ed. (1998) Proteolytic and Cellular Mechanisms in Prohormone and Proprotein Processing, R.G. Landes 5 Knopman, D. (2001) Cerebrospinal fluid beta-amyloid and tau proteins for the diagnosis of Alzheimer disease. Arch. Neurol.
Differential display of complex mixtures A few years ago, two initial publications appeared that clearly demonstrated the capability of MALDI–TOF–MS in semiquantitative analysis of complex peptide mixtures31,32. Although a differential display had been shown only for a few different spectra of rat neurointermediate lobes31 and the haemolymph of Drosophila32, it became clear that an application for other biological sources, such as body fluids, could become possible. With the development of a robust and reproducible separation technology for peptides in complex biological sources by adsorption or affinity purification on a chip21, chromatography yielding a 3D display8 (Fig. 1) and more sophisticated data-mining tools, the comprehensive discovery of human peptides relevant as biomarkers or drug targets has just begun.
58, 373–379 6 Steppan, C.M. et al. (2001) The hormone resistin links obesity to diabetes. Nature 409, 307–312 7 Kennedy, S. (2001) Proteomic profiling from human samples: the body fluid alternative. Toxicol. Lett. 120, 379–384 8 Schulz-Knappe, P. et al. (2001) Peptidomics: the comprehensive analysis of peptides in complex biological mixtures. Comb. Chem. High Throughput Screen. 4, 207–217 9 Walsh, G. (1998) Biopharmaceuticals: Biochemistry and Biotechnology, John Wiley & Sons 10 Lundell, N. (1995) A peptide in a haystack – strategies for a biochromatographer. LC GC 8, 636–647 11 Blackstock, W.P. (2000) Trends in automation and mass spectrometry for proteomics. In Proteomics: A Trends Guide (Blackstock, W. and Mann, M., eds), pp. 12–17, Elsevier 12 Papayannopoulos, I.A. (1995) The interpretation of collisioninduced dissociation tandem mass spectra of peptides. Mass Spectrom.
Prospects The application of peptidomic approaches to analyse human body fluids will complement efforts to study gene expression using proteomic or transcriptomic technologies. It will serve as a tool to study gene expression products in a relevant and complex class of clinically interesting molecules, with the potential for application in clinical analysis and drug development. The knowledge gained from analysis of the peptide content in human body fluids will have a direct impact on the diagnosis of many diseases. In the future, the old paradigm of one disease related to a single gene and diagnosed by a single marker or treated by a single drug, will no longer be valid. The identification of interacting gene expression networks and the measurement of marker patterns must be achieved in order to differentiate between diseases. Using such tools, it will http://trends.com
Rev. 14, 49–73 13 Washburn, M.P. et al. (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242–247 14 Beavis, R.C. and Fenyö, D. (2000) Database searching with massspectrometric information. In Proteomics: A Trends Guide (Blackstock, W. and Mann, M., eds), pp. 22–27, Elsevier 15 Verhaert, P. et al. (2001) Matrix-assisted laser desorption/ionization quadrupole time-of-flight mass spectrometry: an elegant tool for peptidomics. Proteomics 1, 118–131 16 Conrads, T.P. et al. (2000) Utility of accurate mass tags for proteome-wide protein identification. Anal. Chem. 72, 3349–3354 17 Jürgens, M. et al. (1997) Multi-dimensional mapping of human blood peptides by mass spectrometry. J. Biomol. Tech. 9, 24–30 18 Raida, M. et al. (1999) Liquid chromatography and electrospray mass spectrometric mapping of peptides from human plasma filtrate Am. Soc. Mass Spectrom. 10, 45–54
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19 Richter, R. et al. (1999) Composition of the peptide fraction in human blood plasma: data base of circulating human peptides. J. Chromatogr. B 726, 25–35 20 Garnero, P. et al. (2000) Molecular basis and clinical use of
β-2-microglobulin using mass spectrometric immunoassay. Anal. Biochem. 289, 26–35 28 Gobom, J. et al. (2000) Detection and quantification of
biochemical markers of bone, cartilage, and synovium in joint
neurotensin in human brain tissue by MALDI–TOF–MS. Anal. Chem.
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21 Vlahou, A. et al. (2001) Development of a novel proteomic approach
29 Fierens, C. et al. (2000) Quantitative analysis of urinary C-peptide by
for the detection of transitional cell carcinoma of the bladder in
liquid chromatography-tandem mass spectrometry with a stable
urine. Am. J. Pathol. 158, 1491–1502 22 Heine, G. et al. (1997) Mapping of peptides and protein fragments in
isotopically labelled internal standard. J. Chromatogr. A 896, 275–278 30 Castagnola, M. et al. (2001) Determination of the human salivary
human urine using liquid chromatography-mass spectrometry.
peptides histatin 1, 3, 5 and statherin by high-performance liquid
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23 Spahr, C.S. et al. (2001) Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry I. Profiling an unfractionated tryptic digest. Proteomics 1, 93–107 24 Ayad, M. et al. (2000) The association of basic proline-rich peptides from human parotid gland secretions with caries experience. J. Dent. Res. 79, 976–982 25 Britton, J.R. and Kastin, A.J. (1991) Biologically active polypeptides in milk. Am. J. Med. Sci. 301, 124–132 26 Stark, M. et al. (2001) Peptide repertoire of human cerebrospinal fluid: novel proteolytic fragments of neuroendocrine proteins. J. Chromatogr. B 754, 357–367
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27 Tubbs, K.A. et al. (2001) Detection and quantification of
751, 153–160 31 Jimenez, C.R. et al. (1997) Pattern changes of pituitary peptides in rat after salt-loading as detected by means of direct, semiquantitative mass spectrometric profiling. Proc. Natl. Acad. Sci. U. S. A. 94, 9481–9486 32 Uttenweiler-Joseph, S. et al. (1998) Differential display of peptides induced during the immune response of Drosophila: a matrix-assisted laser desorption ionization time-of-flight mass spectrometry study. Proc. Natl. Acad. Sci. U. S. A. 95, 11342–11347 33 König, W. (1993) Peptide and Protein Hormones, VCH Verlagsgesellschaft 34 Cunnane, G. (2001) Amyloid precursors and amyloidosis in inflammatory arthritis. Curr. Opin. Rheumatol. 13, 67–73
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