- Email: [email protected]
Origin of anterior patterning
COMMENT
Origin of anterior patterning
Outlook
Origin of anterior patterning how old is our head? Most animals that display a bilateral symmetry (bilaterians) share homologous regulatory genes involved in head development. Recently, homologues of several of these genes have been cloned from animals that are radially organized, such as coral, sea anemones, jellyfish or hydra (cnidarians). Surprisingly, some of these are expressed apically and/or during apical patterning in hydrozoans, suggesting that head patterning is much older than previously thought. nidarians are simple animals that are composed of two cell layers and that have nerve cells but no organs. They have diverged during evolution before the emergence of bilaterian animals (Fig. 1). Developmental processes leading to head development are considered to be much more recent acquisitions1; so what could be common between the cnidarian ‘head’, a mouth opening surrounded by a ring of tentacles, and that of a vertebrate or an arthropod? Obviously not the level of complexity; however, in each case, active feeding behaviour requires coordination via nerve cells; in cnidarians, this is organized in a nerve net that is localized in the apical region2, whereas in most bilaterians this is organized in a central nervous system (CNS). This role of nerve cells is best demonstrated by nerve-free hydra, which are unable to catch food. Recent molecular analyses have shown that a conserved set of genes is responsible for the establishment of head organizer activity during early embryogenesis across the bilaterians3–6. These regulatory genes belong to three main classes of homeobox genes, the Paired-class (Otx, Goosecoid, Anf/Hesx1), the Antennapedia-class (Emx, Hex/Prh) and the Lim-class (Lim-1), or to the winged-helix class of transcription factors (Axial/Hnf-3b). Later in development, these genes also function in brain patterning and/or neurogenesis. Expression and mutational analysis of these genes together with classical embryological manipulations have shown that, contrary to the classical view according to which head patterning is exclusively dependent on the inducing activity of prechordal mesoderm (as in chick7), the primitive anterior endoderm might actually be the primary site of anterior organizer activity prior to gastrulation in the mouse8 and Xenopus4. Therefore, the ancestral head organizer activity might not have required a competent mesoderm layer, and it is conceivable that diploblasts and triploblasts (animals developed from two- and three-layered embryos, respectively) might share common mechanisms for anterior specification. Recently, homologues of head-specific genes have been cloned from coral, jellyfish, hydractinia and hydra, and, in several cases, they have been shown to be expressed in a apical-specific manner in adult animals and during head formation (Table 1). Thus, these genes are potentially highly informative in terms of both ancestral roles and conserved molecular mechanisms.
C
Paired-like genes, primitive endoderm and head organizer activity A striking common feature of the three paired-like genes involved in early anterior patterning in vertebrates 0168-9525/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(99)01888-0
[Goosecoid (Gsc), orthodenticle (Otx2), Anf/Hesx1] is their sequential expression first, prior to gastrulation, in the anterior endoderm, and subsequently in the anterior neurectoderm3,5,9. In the case of Otx2, these two waves of expression correspond to the induction and to the subsequent specification of forebrain and midbrain territories10. In invertebrates, little is known about head-organizer activity and homologues of the Anf/Hesx1 genes have not been found yet11. However, Otx shows the same kind of biphasic expression pattern in both ascidian12 and amphioxus13, strongly implying a conserved ancestral function in chordate head development. In Drosophila, the gap gene Otx is involved in anterior specification and central nervous system (CNS) development6, whereas Gsc is mainly involved in neurogenesis in post-gastrula embryos14. Both genes are functionally interchangeable between vertebrates and invertebrates9,15. Moreover, otd and Otx2 share some homologous target genes, such as wg/wnt1 or engrailed6,10. Finally, the conservation of Otx function might also extend to the leech, where it is largely expressed in the head region16 and to planaria17,18. The two planarian Otx genes define distinct domains in the adult
TABLE 1. Cnidarian homologues of genes involved in organizer activity in triploblastic animals Cnidarian genes Related triploblastic genes Paired-class Paired-like Prdl-a Prdl-b Otx Pax-type Pax-A Pax-B Pax-C Antp-class Cn-emx cnox-1 Winged-helix Budhead
T-box HyBra1
Expression in cnidarians
Ref.
Adult head
Apical patterning
Arx Arx Otx
yes (nerve cells) no no
yes (endoderm) no cell movement
21 21 19
Poxneuro Pax-2, Pax-5, Pax-8 Eyegone, Pax-4, Pax-6
nd nd nd
nd nd nd
20 20 22
empty spiracle, emx Hox-related (PG1)
yes nd
nd yes
36 37
Forkhead, axial, Hnf-3 HFH4, XFKH5, Hyfkh2 FD5, lin-31, FKH4, Hyfkh3
yes
yes (endoderm)
38
nd nd
nd nd
38 38
Brachyurya
yes
yes (endoderm)
39
a
The Brachyury homologue is implicated in early apical patterning in hydra. Abbreviation: nd, not determined.
TIG January 2000, volume 16, No. 1
1
Outlook
COMMENT
Origin of anterior patterning
FIGURE 1. A possible view of animal evolution Triploblastic (bilaterian)
– 300 MY
Vertebrata (fish, chick, Xenopus, mouse, human)
Cephalochordata (amphioxus)
Deuterostomes
Urochordata (ascidia)
Arthropoda (fly)
Nematoda (round worms)
Mollusca (snail)
Brachipoda (tentaculates)
Annelida (leech)
Nemertea (ribbon worms)
Protostomes Lophotrochoz. Ecdysoz.
Platyhelminthes (flat worms)
Ctenophora (comb jellyfish)
Hydrozoan (hydra)
Scyphozoan (jelly fish)
Anthozoans (corals, sea anemone)
Today
Porifera (sponges)
Coelenterates Cnidaria
Echinoderms (sea urchin)
Diploblastic
– 500 MY
CNS (cephalic ganglion eyes, neural tube) – 700 MY
Nerve net, sensory organs oral–aboral polarity (mo)
Active feeding behaviour
Nerve cells – 900 MY Plants Fungi (yeast)
Common ancestor (protist) Brigitte Galliot brigitte.galliot@ zoo.unige.ch David Miller* david.miller@ jcu.edu.au Department of Zoology and Animal Biology, University of Geneva, Geneva, Switzerland. *Department of Biochemistry and Molecular Biology, James Cook University, Townsville, Australia. 2
trends in Genetics
The tree is based on 18S rRNA phylogenies30–32 confirmed by analysis of homeodomain sequences concerning the triploblastic animals33. Diploblastics develop from two-cell layered embryos while triploblastics develop from three. The monophyly of cnidarians as well as the basal position of Porifera among diploblastic animals is well established30,34. Analysis of 18 protein-coding gene loci estimated that protostomes diverged from deuterostomes about 670 million years (MY) ago35. Early evolutionary steps of head development are indicated (mo: mouth opening); animal species displaying nerve cells but no polarity are not known2.
brain18 and, in addition, are specifically up-regulated during anterior regeneration17,18. These results support the view that deuterostomes and protostomes share common regulatory molecular mechanisms for their anterior specification and brain patterning. In hydra, an Otx homologue gene appears to have a role in the cell movements leading to the formation of new axes19. Hydra budding and regeneration offer an amenable model system to investigate head organizer activity. Among the seven distinct cnidarian Paired-class genes TIG January 2000, volume 16, No. 1
identified19–22, the hydra paired-like gene prdl-a, shows an expression pattern reminiscent of that observed for Otx, Gsc and Anf/Hesx1 in vertebrates. During regeneration, prdl-a expression is first detected in the apical endoderm at the place and at the time to which head organizer activity has been mapped by transplantation experiments; it is subsequently expressed in the adjacent ectoderm21. This sequential expression pattern suggests that, in cnidarians as in most chordates, early anterior endodermal cells are responsible for head organizer activity by signalling to
COMMENT
Origin of anterior patterning
Outlook
FIGURE 2. Co-evolution of homeodomain-containing proteins and head-organizer activity Dictyostelium
Evolutionary time
Porifera (Sponges)
Cnidaria (Coral, jellyfish, hydra))
Pax-2/5/8
Pax-A like 5
Planaria, Annelida, Nematoda, Arthropoda, Echinoderms, Chordata
Pax-B
Pax 2/5/8
Pax-A
Pax neuro Pax 1/9, Pax 3, Pax 7 Eyg, Pax 4, Pax 6
Pax-C
paired-type HP S50 4 1
Wariai Q50
paired-type HP Q50
2
Lim-class Q50
prdl-a prdl-b ? ? ?
Aristaless Orthopedia smox3, Prx1–2 Unc-4, Rax/rx Chx10, Cart1 Siamois, Mix Anf/Hesx1 3
Otx-type HP Q50 Hex Emx Para-Hox, Hox
Antp-class Q50
Otx
Hbx4 Otx Gsc Ptx CNS patterning
Head development Nerve cell differentiation Inductive interactions Cell differentiation, polarity Homeodomain (HD)
Conserved intron within HD
Paired domain (PD)
Octapeptide (OP)
OAR or C-peptide trends in Genetics
Head organizer activity was established in diploblastic phyla at the time the Antp-class and Paired-class homeobox gene families diversified. Several Antp-class and most Paired-class homeoproteins (HP) are involved in anterior specification, central nervous system (CNS) patterning and/or neurogenesis in a conserved manner. Their proposed history is deduced from the analysis of their structure and homeodomain (HD) sequences. Paired-class HDs share six diagnostic residues (P26, D27, E32, R44, Q46, A54), some of which can be traced back to the Lim-class (P26, D27, E32, Q46, A54) and the Dictyostelium Wariai HDs (P26, Q46, A54)11,29 . Paired-like HPs are defined by a typical paired-class HD but no paired-domain (PD). Residue 50 of the HD is a key residue for DNA-binding specificity. A possible evolutionary scenario is proposed whereby an ancestral paired-like gene encoding a Q50 HD and OAR domain (step 1) arose from a common ancestor of Lim-, Antp- and Paired-class genes. Subsequently Otx-type genes resulted from the transformation of residue 50 (Q50➔K50, step 2), some of these sequences having more recently reverted (K50➔Q50, step 3, see Hbx4 and Anf ). Pax genes also arose from the transformation of residue 50 of the HD (Q50➔S50, step 4, no extant representative identified to date) and the fusion of this S50 HD with an ancestral PD (Pax-A like, step 5). HD encoded by the Pax-2/5/8 genes is always partial, suggesting it degenerated very early or was acquired independently (as presented here). All the genes mentioned on the right-hand side are involved in further development of the head or CNS in triploblastics.
the overlying ectodermal layer. Strikingly, regulatory genes that are expressed during early head regeneration, display transcripts located in the endodermal cells of the stump (Table 1 and B. Galliot, unpublished). In the mouse, the endodermal Hesx1- and Otx2expressing cells induce corresponding Hesx1 or Otx2 expression in the overlying ectoderm8,10. Although a similar interaction has yet to be demonstrated for hydra prdl-a, these morphogenetic interactions from endoderm to
ectoderm could eventually turn out to be homologous. In hydra, endodermal cells of the bud or the regenerating tip transiently participate in head activation, later differentiating into gland cells or epithelial cells, assuming a gastric function. In the mouse embryo, the primitive endoderm makes no contribution to embryonic tissues but, once its organizer role has been achieved, is incorporated into extraembryonic tissues. In both cases, the overlying ectodermal layer will give rise, although not exclusively, to TIG January 2000, volume 16, No. 1
3
Outlook
COMMENT
Origin of anterior patterning
neural tissue: nerve cells organized as a net in hydra and as a brain in the case of vertebrates. The signalling role of the mesoderm during gastrulation in higher animals suggests that this layer was recruited from the endoderm, later in evolution. Accordingly, in zebrafish both layers probably originate from a common pathway23.
When did head organizer activity appear during evolution? As discussed above, active feeding behaviour provides a common definition for the animal head: this behaviour relies on differentiation of both nerve cells and a localized pole in the animal. Whereas under this definition, cnidarians and bilaterians have a head, sponges do not: their feeding is the result of a passive filtration process that is not localized at one pole of the animal and that does not involve nerve cells, which are absent in sponges (Porifera, Fig. 1). Two major evolutionary steps therefore separate the Porifera and Cnidaria: the differentiation of nerve cells, permitting rapid responses to external stimulus, and the determination of an active oral pole, enabling the animal to capitalize upon its responsiveness. The second of these steps (apical differentiation) was probably permitted by the first (nerve cell differentiation): concentration of nerve cells at one pole of the animal allowed great increases in the efficiency of feeding behaviour. By participating in morphogenetic interactions from endoderm to ectoderm, Paired-like genes might have been key players in the early evolution of animal head (Fig. 2). In bilaterians, the Pax-2/5/8 class of genes specify cell fate in the CNS in a conserved manner24,25 but do not participate in early anterior patterning processes26. The recent identification of a sponge gene related to this class27 implies that this Pax class predates the divergence of the Porifera as well as the evolution of nerve cells. Thus, its function in neurogenesis might represent a more recent recruitment, possibly predating the cnidarian divergence.
References 1 Gans, C. and Northcutt, R.G. (1983) Neural crest and the origin of vertebrates: a new head. Science 220, 268–274 2 Grimmelikhuijzen, C.J.P. and Westfall, J.A. (1995) The nervous system of Cnidarians. In The Nervous Systems of Invertebrates: An Evolutionary and Comparative Approach (Breidbach, O. and Kutsch, W., eds), pp. 7–24, Birkhaüser Verlag 3 Bally-Cuif, L. and Boncinelli, E. (1997) Transcription factors and head formation in vertebrates. BioEssays 19, 127–135 4 Bouwmeester, T. and Leyns, L. (1997) Vertebrate head induction by anterior primitive endoderm. BioEssays 19, 855–863 5 Beddington, R.S. and Robertson, E.J. (1998) Anterior patterning in mouse. Trends Genet. 14, 277–284 6 Hartmann, B. and Reichert, H. (1998) The genetics of embryonic brain development in Drosophila. Mol. Cell. Neurosci. 12, 194–205 7 Knoetgen, H. et al. (1999) Head induction in the chick by primitive endoderm of mammalian, but not avian origin. Development 126, 815–825 8 Thomas, P. and Beddington, R. (1996) Anterior primitive endoderm may be responsible for patterning the anterior neural plate in the mouse embryo. Curr. Biol. 6, 1487–1496 9 Acampora, D. et al. (1999) Otx genes and the genetic control of brain morphogenesis. Mol. Cell. Neurosci. 13, 1–8 10 Rhinn, M. et al. (1998) Sequential roles for otx2 in visceral endoderm and neuroectoderm for forebrain and midbrain induction and specification. Development 125, 845–856 11 Galliot, B. et al. (1999) Evolution of homeobox genes: Q50 Paired-like genes founded the paired class. Dev. Genes Evol. 209, 186–197 12 Wada, S. et al. (1996) Hroth an orthodenticle-related homeobox gene of the ascidian, Halocynthia roretzi: its expression and putative roles in the axis formation during
4
If this possibility is verified in cnidarians20, then the recruitment of the Pax-2/5/8 gene for neurogenesis could be one of the earliest markers for the origin of neurogenesis, while the Paired-like family may have been more specifically recruited for the next step, i.e. apical organizer activity. Members of the Forkhead, Paired-like, emx and T-box gene families have been implicated in apical patterning in cnidarians but have not so far been identified in sponges which might mean that they appeared during evolution and/or became stabilized at the time when a rudimentary head developed. LIM-class genes, which are involved in anterior organizer activity in chordates3–5,12 and neurogenesis in Drosophila28, predate Paired-like genes in our analysis11. Thus, cnidarian Lim-related genes, not yet identified, might have similar functions. Finally, some of these regulatory developmental pathways might predate true multicellularity: the Dictyostelium gene, Wariai, is involved in antero–posterior differentiation through a non-autonomous mechanism29, and encodes a Q50 homeodomain (see Fig. 2) that has three of the six paired-class diagnostic residues. Wariai could thus represent the common ancestor of both Lim and Paired-like homeobox genes. In conclusion, this hypothesis – that some of the developmental pathways leading to anterior patterning in bilaterians were already present in their common ancestor – starts to find some echo in the rudimentary cnidarian head. We propose that, as most of the Paired-like genes contribute to head organizer activity and/or brain organogenesis or neurogenesis, their divergence along evolution accompanied and facilitated the process of head development.
Acknowledgements We thank C. Desplan for critical reading of the manuscript. The work in our laboratories is supported by the Swiss National Foundation, Canton of Geneva, Australian Research Council and James Cook University.
embryogenesis. Mech. Dev. 60, 59–71 13 Williams, N. and Holland, P. (1996) Old head on young shoulders. Nature 383, 490 14 Hahn, M. and Jackle, H. (1996) Drosophila goosecoid participates in neural development but not in body axis formation. EMBO J. 15, 3077–3084 15 Goriely, A. et al. (1996) A functional homologue of goosecoid in Drosophila. Development 122, 1641–1650 16 Bruce, A.E. and Shankland, M. (1998) Expression of the head gene Lox22-Otx in the leech Helobdella and the origin of the bilaterian body plan. Dev. Biol. 201, 101–112 17 Stornaiuolo, A. et al. (1998) A homeobox gene of the orthodenticle family is involved in antero–posterior patterning of regenerating planarians. Int. J. Dev. Biol. 42, 1153–1158 18 Umesono, Y. et al. (1999) Distinct structural domains in the planarian brain defined by the expression of evolutionarily conserved homeobox genes. Dev. Genes Evol. 209, 31–39 19 Smith, K.M. et al. (1999) CnOtx, a member of the otx gene family, has a role in cell movement in hydra. Dev. Biol. 212, 392–404 20 Sun, H. et al. (1997) Evolution of paired domains: isolation and sequencing of jellyfish and hydra Pax genes related to Pax-5 and Pax-6. Proc. Natl. Acad. Sci. U. S. A. 94, 5156–5161 21 Gauchat, D. et al. (1998) prdl-a, a gene marker for hydra apical differentiation related to triploblastic paired-like head-specific genes. Development 125, 1637–1645 22 Catmull, J. et al. (1998) Pax-6 origins – implications from the structure of two coral Pax genes. Dev. Genes Evol. 208, 352–356 23 Warga, R.M. and Nusslein-Volhard, C. (1999) Origin and development of the zebrafish endoderm. Development 126, 827–838 24 Fu, W. and Noll, M. (1997) The Pax2 homolog sparkling is required for development of cone and pigment cells in the Drosophila eye. Genes Dev. 11, 2066–2078
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25 Wada, H. et al. (1998) Tripartite organization of the ancestral chordate brain and the antiquity of placodes: insights from ascidian Pax-2/5/8, Hox and Otx genes. Development 125, 1113–1122 26 Dahl, E. et al. (1997) Pax genes and organogenesis. BioEssays 19, 755–765 27 Hoshiyama, D. et al. (1998) Sponge pax cDNA related to pax-2/5/8 and ancient gene duplications in the pax family. J. Mol. Evol. 47, 640–648 28 Lundgren, S.E. et al. (1995) Control of neuronal pathway selection by the Drosophila LIM homeodomain gene apterous. Development 121, 1769–1773 29 Han, Z. and Firtel, R.A. (1998) The homeobox-containing gene Wariai regulates anterior–posterior patterning and cell-type homeostasis in Dictyostelium. Development 125, 313–325 30 Wainright, P.O. et al. (1993) Monophyletic origins of the metazoa: an evolutionary link with fungi. Science 260, 340–342 31 Aguinaldo, A.M. et al. (1997) Evidence for a clade of nematodes, arthropods and other moulting animals. Nature 387, 489–493 32 Veuthey, A.L. and Bittar, G. (1998) Phylogenetic relationships of fungi, plantae, and animalia inferred from homologous comparison of ribosomal proteins. J. Mol. Evol. 47, 81–92 33 de Rosa, R. et al. (1999) Hox genes in brachiopods and priapulids and protostome evolution. Nature 399, 772–776 34 Odorico, D.M. and Miller, D.J. (1997) Internal and external relationships of the Cnidaria: implications of primary and predicted secondary structure of the 59-end of the 23S-like rDNA. Proc. R. Soc. London B Biol. Sci. 264, 77–82 35 Ayala, F.J. and Rzhetsky, A. (1998) Origin of the metazoan phyla: molecular clocks confirm paleontological estimates. Proc. Natl. Acad. Sci. U. S. A. 95, 606–611 36 Mokady, O. et al. (1998) Over one-half billion years of head conservation? Expression of an ems class gene in Hydractinia
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symbiolongicarpus (Cnidaria: Hydrozoa). Proc. Natl. Acad. Sci. U. S. A. 95, 3673–3678 37 Schummer, M. et al. (1992) HOM/HOX homeobox genes are present in hydra (Chlorohydra viridissima) and are
differentially expressed during regeneration. EMBO J. 11, 1815–1823 38 Martinez, D.E. et al. (1997) Budhead, a forkhead/HNF3 homologue, is expressed during axis formation and head
Outlook
specification in hydra. Dev. Biol. 192, 523–536 39 Technau, U. and Bode, H.R. (1999) HyBra1, a Brachyury homologue, acts during head formation in Hydra. Development 126, 999–1010
Mass spectrometry from genomics to proteomics Large-scale DNA sequencing has stimulated the development of proteomics by providing a sequence infrastructure for protein analysis. Rapid and automated protein identification can be achieved by searching protein and nucleotide sequence databases directly with data generated by mass spectrometry. A highthroughput and large-scale approach to identifying proteins has been the result. These technological changes have advanced protein expression studies and the identification of proteins in complexes, two types of studies that are essential in deciphering the networks of proteins that are involved in biological processes. he elucidation of an organism’s genome is the first and important step towards understanding its biology, and the data created by whole-genome sequencing have significant benefits in fields outside those of genomics and bioinformatics. One area to benefit is that of proteomics. The term proteomics, or more appropriately functional proteomics, describes the ability to apply global (proteomewide or system-wide) experimental approaches to assess protein function. Proteomics has emerged as a new experimental approach in part because mass spectrometry has simplified protein analysis and characterization, and several important and recent innovations have extended the capability of mass spectrometry.
T
Mass spectrometry of biological molecules Mass spectrometers consist of three essential parts (Fig. 1). The first, an ionization source, converts molecules into gas-phase ions. Once ions are created, individual mass-tocharge ratios (m/z; see Box 1) are separated by a second device, a mass analyzer, and transferred to the third, an ion detector. A mass analyzer uses a physical property [e.g. electric or magnetic fields, or time-of-flight (TOF)] to separate ions of a particular m/z value that then strike the ion detector. The magnitude of the current that is produced at the detector as a function of time (i.e. the physical field in the mass analyzer is changed as a function of time) is used to determine the m/z value of the ion. Although mass analyzers are an important (and continually improving) component of mass spectrometers and determine critical performance characteristics, an important innovation for proteomics has been the development of two robust techniques to create ions of large molecules. Matrix-assisted laser desorption ionization (MALDI) creates ions by excitation of molecules that are isolated from the energy of the laser by an energy absorbing matrix. The laser energy strikes the crystalline matrix to cause rapid excitation of the matrix and subsequent ejection of matrix and analyte ions into the gas-phase. Electrospray ionization (ESI) creates ions by application of a potential to a flowing liquid causing the liquid 0168-9525/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(99)01879-X
to charge and subsequently spray. The electrospray creates very small droplets of solvent-containing analyte. Solvent is removed as the droplets enter the mass spectrometer by heat or some other form of energy (e.g. energetic collisions with a gas), and multiply-charged ions are formed in the process. The detection limits that can be achieved with ESI have improved with a reduction in the flow rates1. These ionization techniques have stimulated developments in mass spectrometers to enhance the production of two different types of information. The first type of information is the accurate measurement of molecular weight. To measure molecular weight to the low ppm level, MALDI is used typically in conjunction with TOF mass analyzers. The second type of information, produced by tandem mass spectrometers (MS/MS), is diagnostic of amino acid sequence (Fig. 1b). Many types of MS/MS have been developed2, and new innovations allow greater automation and efficiency in data acquisition. Data can be generated in a data-dependent manner through interaction of the m/z data in each scan with a computer program to control the type of experiment performed3. For example, a scan of the mass range can reveal the presence of several ions above a preset ion-abundance threshold. The computer can signal the instrument to perform tandem mass spectrometry on each of the ions, thus improving the efficiency of data acquisition, particularly during separations when ions appear for only a brief period of time.
Identifying proteins using mass spectrometry data and database searching Mass spectrometers are capable of generating data quickly and thus have a great potential for high-throughput analysis. An essential component to achieving greater throughput is simplifying data analysis. There is a direct relationship between mass spectrometry data and amino acid sequences. Peptide molecular weight measurements are predictive of amino acid composition, and peptide fragmentation information (as described in the glossary) relates to amino acid sequence. Both types of information can be correlated to protein sequences in the database. A single peptide TIG January 2000, volume 16, No. 1
John R. Yates, III jyates@ u.washington.edu Department of Molecular Biotechnology, University of Washington, Seattle, WA 98195-7730, USA. 5
Origin of anterior patterning
Outlook
Origin of anterior patterning how old is our head? Most animals that display a bilateral symmetry (bilaterians) share homologous regulatory genes involved in head development. Recently, homologues of several of these genes have been cloned from animals that are radially organized, such as coral, sea anemones, jellyfish or hydra (cnidarians). Surprisingly, some of these are expressed apically and/or during apical patterning in hydrozoans, suggesting that head patterning is much older than previously thought. nidarians are simple animals that are composed of two cell layers and that have nerve cells but no organs. They have diverged during evolution before the emergence of bilaterian animals (Fig. 1). Developmental processes leading to head development are considered to be much more recent acquisitions1; so what could be common between the cnidarian ‘head’, a mouth opening surrounded by a ring of tentacles, and that of a vertebrate or an arthropod? Obviously not the level of complexity; however, in each case, active feeding behaviour requires coordination via nerve cells; in cnidarians, this is organized in a nerve net that is localized in the apical region2, whereas in most bilaterians this is organized in a central nervous system (CNS). This role of nerve cells is best demonstrated by nerve-free hydra, which are unable to catch food. Recent molecular analyses have shown that a conserved set of genes is responsible for the establishment of head organizer activity during early embryogenesis across the bilaterians3–6. These regulatory genes belong to three main classes of homeobox genes, the Paired-class (Otx, Goosecoid, Anf/Hesx1), the Antennapedia-class (Emx, Hex/Prh) and the Lim-class (Lim-1), or to the winged-helix class of transcription factors (Axial/Hnf-3b). Later in development, these genes also function in brain patterning and/or neurogenesis. Expression and mutational analysis of these genes together with classical embryological manipulations have shown that, contrary to the classical view according to which head patterning is exclusively dependent on the inducing activity of prechordal mesoderm (as in chick7), the primitive anterior endoderm might actually be the primary site of anterior organizer activity prior to gastrulation in the mouse8 and Xenopus4. Therefore, the ancestral head organizer activity might not have required a competent mesoderm layer, and it is conceivable that diploblasts and triploblasts (animals developed from two- and three-layered embryos, respectively) might share common mechanisms for anterior specification. Recently, homologues of head-specific genes have been cloned from coral, jellyfish, hydractinia and hydra, and, in several cases, they have been shown to be expressed in a apical-specific manner in adult animals and during head formation (Table 1). Thus, these genes are potentially highly informative in terms of both ancestral roles and conserved molecular mechanisms.
C
Paired-like genes, primitive endoderm and head organizer activity A striking common feature of the three paired-like genes involved in early anterior patterning in vertebrates 0168-9525/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(99)01888-0
[Goosecoid (Gsc), orthodenticle (Otx2), Anf/Hesx1] is their sequential expression first, prior to gastrulation, in the anterior endoderm, and subsequently in the anterior neurectoderm3,5,9. In the case of Otx2, these two waves of expression correspond to the induction and to the subsequent specification of forebrain and midbrain territories10. In invertebrates, little is known about head-organizer activity and homologues of the Anf/Hesx1 genes have not been found yet11. However, Otx shows the same kind of biphasic expression pattern in both ascidian12 and amphioxus13, strongly implying a conserved ancestral function in chordate head development. In Drosophila, the gap gene Otx is involved in anterior specification and central nervous system (CNS) development6, whereas Gsc is mainly involved in neurogenesis in post-gastrula embryos14. Both genes are functionally interchangeable between vertebrates and invertebrates9,15. Moreover, otd and Otx2 share some homologous target genes, such as wg/wnt1 or engrailed6,10. Finally, the conservation of Otx function might also extend to the leech, where it is largely expressed in the head region16 and to planaria17,18. The two planarian Otx genes define distinct domains in the adult
TABLE 1. Cnidarian homologues of genes involved in organizer activity in triploblastic animals Cnidarian genes Related triploblastic genes Paired-class Paired-like Prdl-a Prdl-b Otx Pax-type Pax-A Pax-B Pax-C Antp-class Cn-emx cnox-1 Winged-helix Budhead
T-box HyBra1
Expression in cnidarians
Ref.
Adult head
Apical patterning
Arx Arx Otx
yes (nerve cells) no no
yes (endoderm) no cell movement
21 21 19
Poxneuro Pax-2, Pax-5, Pax-8 Eyegone, Pax-4, Pax-6
nd nd nd
nd nd nd
20 20 22
empty spiracle, emx Hox-related (PG1)
yes nd
nd yes
36 37
Forkhead, axial, Hnf-3 HFH4, XFKH5, Hyfkh2 FD5, lin-31, FKH4, Hyfkh3
yes
yes (endoderm)
38
nd nd
nd nd
38 38
Brachyurya
yes
yes (endoderm)
39
a
The Brachyury homologue is implicated in early apical patterning in hydra. Abbreviation: nd, not determined.
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Origin of anterior patterning
FIGURE 1. A possible view of animal evolution Triploblastic (bilaterian)
– 300 MY
Vertebrata (fish, chick, Xenopus, mouse, human)
Cephalochordata (amphioxus)
Deuterostomes
Urochordata (ascidia)
Arthropoda (fly)
Nematoda (round worms)
Mollusca (snail)
Brachipoda (tentaculates)
Annelida (leech)
Nemertea (ribbon worms)
Protostomes Lophotrochoz. Ecdysoz.
Platyhelminthes (flat worms)
Ctenophora (comb jellyfish)
Hydrozoan (hydra)
Scyphozoan (jelly fish)
Anthozoans (corals, sea anemone)
Today
Porifera (sponges)
Coelenterates Cnidaria
Echinoderms (sea urchin)
Diploblastic
– 500 MY
CNS (cephalic ganglion eyes, neural tube) – 700 MY
Nerve net, sensory organs oral–aboral polarity (mo)
Active feeding behaviour
Nerve cells – 900 MY Plants Fungi (yeast)
Common ancestor (protist) Brigitte Galliot brigitte.galliot@ zoo.unige.ch David Miller* david.miller@ jcu.edu.au Department of Zoology and Animal Biology, University of Geneva, Geneva, Switzerland. *Department of Biochemistry and Molecular Biology, James Cook University, Townsville, Australia. 2
trends in Genetics
The tree is based on 18S rRNA phylogenies30–32 confirmed by analysis of homeodomain sequences concerning the triploblastic animals33. Diploblastics develop from two-cell layered embryos while triploblastics develop from three. The monophyly of cnidarians as well as the basal position of Porifera among diploblastic animals is well established30,34. Analysis of 18 protein-coding gene loci estimated that protostomes diverged from deuterostomes about 670 million years (MY) ago35. Early evolutionary steps of head development are indicated (mo: mouth opening); animal species displaying nerve cells but no polarity are not known2.
brain18 and, in addition, are specifically up-regulated during anterior regeneration17,18. These results support the view that deuterostomes and protostomes share common regulatory molecular mechanisms for their anterior specification and brain patterning. In hydra, an Otx homologue gene appears to have a role in the cell movements leading to the formation of new axes19. Hydra budding and regeneration offer an amenable model system to investigate head organizer activity. Among the seven distinct cnidarian Paired-class genes TIG January 2000, volume 16, No. 1
identified19–22, the hydra paired-like gene prdl-a, shows an expression pattern reminiscent of that observed for Otx, Gsc and Anf/Hesx1 in vertebrates. During regeneration, prdl-a expression is first detected in the apical endoderm at the place and at the time to which head organizer activity has been mapped by transplantation experiments; it is subsequently expressed in the adjacent ectoderm21. This sequential expression pattern suggests that, in cnidarians as in most chordates, early anterior endodermal cells are responsible for head organizer activity by signalling to
COMMENT
Origin of anterior patterning
Outlook
FIGURE 2. Co-evolution of homeodomain-containing proteins and head-organizer activity Dictyostelium
Evolutionary time
Porifera (Sponges)
Cnidaria (Coral, jellyfish, hydra))
Pax-2/5/8
Pax-A like 5
Planaria, Annelida, Nematoda, Arthropoda, Echinoderms, Chordata
Pax-B
Pax 2/5/8
Pax-A
Pax neuro Pax 1/9, Pax 3, Pax 7 Eyg, Pax 4, Pax 6
Pax-C
paired-type HP S50 4 1
Wariai Q50
paired-type HP Q50
2
Lim-class Q50
prdl-a prdl-b ? ? ?
Aristaless Orthopedia smox3, Prx1–2 Unc-4, Rax/rx Chx10, Cart1 Siamois, Mix Anf/Hesx1 3
Otx-type HP Q50 Hex Emx Para-Hox, Hox
Antp-class Q50
Otx
Hbx4 Otx Gsc Ptx CNS patterning
Head development Nerve cell differentiation Inductive interactions Cell differentiation, polarity Homeodomain (HD)
Conserved intron within HD
Paired domain (PD)
Octapeptide (OP)
OAR or C-peptide trends in Genetics
Head organizer activity was established in diploblastic phyla at the time the Antp-class and Paired-class homeobox gene families diversified. Several Antp-class and most Paired-class homeoproteins (HP) are involved in anterior specification, central nervous system (CNS) patterning and/or neurogenesis in a conserved manner. Their proposed history is deduced from the analysis of their structure and homeodomain (HD) sequences. Paired-class HDs share six diagnostic residues (P26, D27, E32, R44, Q46, A54), some of which can be traced back to the Lim-class (P26, D27, E32, Q46, A54) and the Dictyostelium Wariai HDs (P26, Q46, A54)11,29 . Paired-like HPs are defined by a typical paired-class HD but no paired-domain (PD). Residue 50 of the HD is a key residue for DNA-binding specificity. A possible evolutionary scenario is proposed whereby an ancestral paired-like gene encoding a Q50 HD and OAR domain (step 1) arose from a common ancestor of Lim-, Antp- and Paired-class genes. Subsequently Otx-type genes resulted from the transformation of residue 50 (Q50➔K50, step 2), some of these sequences having more recently reverted (K50➔Q50, step 3, see Hbx4 and Anf ). Pax genes also arose from the transformation of residue 50 of the HD (Q50➔S50, step 4, no extant representative identified to date) and the fusion of this S50 HD with an ancestral PD (Pax-A like, step 5). HD encoded by the Pax-2/5/8 genes is always partial, suggesting it degenerated very early or was acquired independently (as presented here). All the genes mentioned on the right-hand side are involved in further development of the head or CNS in triploblastics.
the overlying ectodermal layer. Strikingly, regulatory genes that are expressed during early head regeneration, display transcripts located in the endodermal cells of the stump (Table 1 and B. Galliot, unpublished). In the mouse, the endodermal Hesx1- and Otx2expressing cells induce corresponding Hesx1 or Otx2 expression in the overlying ectoderm8,10. Although a similar interaction has yet to be demonstrated for hydra prdl-a, these morphogenetic interactions from endoderm to
ectoderm could eventually turn out to be homologous. In hydra, endodermal cells of the bud or the regenerating tip transiently participate in head activation, later differentiating into gland cells or epithelial cells, assuming a gastric function. In the mouse embryo, the primitive endoderm makes no contribution to embryonic tissues but, once its organizer role has been achieved, is incorporated into extraembryonic tissues. In both cases, the overlying ectodermal layer will give rise, although not exclusively, to TIG January 2000, volume 16, No. 1
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Origin of anterior patterning
neural tissue: nerve cells organized as a net in hydra and as a brain in the case of vertebrates. The signalling role of the mesoderm during gastrulation in higher animals suggests that this layer was recruited from the endoderm, later in evolution. Accordingly, in zebrafish both layers probably originate from a common pathway23.
When did head organizer activity appear during evolution? As discussed above, active feeding behaviour provides a common definition for the animal head: this behaviour relies on differentiation of both nerve cells and a localized pole in the animal. Whereas under this definition, cnidarians and bilaterians have a head, sponges do not: their feeding is the result of a passive filtration process that is not localized at one pole of the animal and that does not involve nerve cells, which are absent in sponges (Porifera, Fig. 1). Two major evolutionary steps therefore separate the Porifera and Cnidaria: the differentiation of nerve cells, permitting rapid responses to external stimulus, and the determination of an active oral pole, enabling the animal to capitalize upon its responsiveness. The second of these steps (apical differentiation) was probably permitted by the first (nerve cell differentiation): concentration of nerve cells at one pole of the animal allowed great increases in the efficiency of feeding behaviour. By participating in morphogenetic interactions from endoderm to ectoderm, Paired-like genes might have been key players in the early evolution of animal head (Fig. 2). In bilaterians, the Pax-2/5/8 class of genes specify cell fate in the CNS in a conserved manner24,25 but do not participate in early anterior patterning processes26. The recent identification of a sponge gene related to this class27 implies that this Pax class predates the divergence of the Porifera as well as the evolution of nerve cells. Thus, its function in neurogenesis might represent a more recent recruitment, possibly predating the cnidarian divergence.
References 1 Gans, C. and Northcutt, R.G. (1983) Neural crest and the origin of vertebrates: a new head. Science 220, 268–274 2 Grimmelikhuijzen, C.J.P. and Westfall, J.A. (1995) The nervous system of Cnidarians. In The Nervous Systems of Invertebrates: An Evolutionary and Comparative Approach (Breidbach, O. and Kutsch, W., eds), pp. 7–24, Birkhaüser Verlag 3 Bally-Cuif, L. and Boncinelli, E. (1997) Transcription factors and head formation in vertebrates. BioEssays 19, 127–135 4 Bouwmeester, T. and Leyns, L. (1997) Vertebrate head induction by anterior primitive endoderm. BioEssays 19, 855–863 5 Beddington, R.S. and Robertson, E.J. (1998) Anterior patterning in mouse. Trends Genet. 14, 277–284 6 Hartmann, B. and Reichert, H. (1998) The genetics of embryonic brain development in Drosophila. Mol. Cell. Neurosci. 12, 194–205 7 Knoetgen, H. et al. (1999) Head induction in the chick by primitive endoderm of mammalian, but not avian origin. Development 126, 815–825 8 Thomas, P. and Beddington, R. (1996) Anterior primitive endoderm may be responsible for patterning the anterior neural plate in the mouse embryo. Curr. Biol. 6, 1487–1496 9 Acampora, D. et al. (1999) Otx genes and the genetic control of brain morphogenesis. Mol. Cell. Neurosci. 13, 1–8 10 Rhinn, M. et al. (1998) Sequential roles for otx2 in visceral endoderm and neuroectoderm for forebrain and midbrain induction and specification. Development 125, 845–856 11 Galliot, B. et al. (1999) Evolution of homeobox genes: Q50 Paired-like genes founded the paired class. Dev. Genes Evol. 209, 186–197 12 Wada, S. et al. (1996) Hroth an orthodenticle-related homeobox gene of the ascidian, Halocynthia roretzi: its expression and putative roles in the axis formation during
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If this possibility is verified in cnidarians20, then the recruitment of the Pax-2/5/8 gene for neurogenesis could be one of the earliest markers for the origin of neurogenesis, while the Paired-like family may have been more specifically recruited for the next step, i.e. apical organizer activity. Members of the Forkhead, Paired-like, emx and T-box gene families have been implicated in apical patterning in cnidarians but have not so far been identified in sponges which might mean that they appeared during evolution and/or became stabilized at the time when a rudimentary head developed. LIM-class genes, which are involved in anterior organizer activity in chordates3–5,12 and neurogenesis in Drosophila28, predate Paired-like genes in our analysis11. Thus, cnidarian Lim-related genes, not yet identified, might have similar functions. Finally, some of these regulatory developmental pathways might predate true multicellularity: the Dictyostelium gene, Wariai, is involved in antero–posterior differentiation through a non-autonomous mechanism29, and encodes a Q50 homeodomain (see Fig. 2) that has three of the six paired-class diagnostic residues. Wariai could thus represent the common ancestor of both Lim and Paired-like homeobox genes. In conclusion, this hypothesis – that some of the developmental pathways leading to anterior patterning in bilaterians were already present in their common ancestor – starts to find some echo in the rudimentary cnidarian head. We propose that, as most of the Paired-like genes contribute to head organizer activity and/or brain organogenesis or neurogenesis, their divergence along evolution accompanied and facilitated the process of head development.
Acknowledgements We thank C. Desplan for critical reading of the manuscript. The work in our laboratories is supported by the Swiss National Foundation, Canton of Geneva, Australian Research Council and James Cook University.
embryogenesis. Mech. Dev. 60, 59–71 13 Williams, N. and Holland, P. (1996) Old head on young shoulders. Nature 383, 490 14 Hahn, M. and Jackle, H. (1996) Drosophila goosecoid participates in neural development but not in body axis formation. EMBO J. 15, 3077–3084 15 Goriely, A. et al. (1996) A functional homologue of goosecoid in Drosophila. Development 122, 1641–1650 16 Bruce, A.E. and Shankland, M. (1998) Expression of the head gene Lox22-Otx in the leech Helobdella and the origin of the bilaterian body plan. Dev. Biol. 201, 101–112 17 Stornaiuolo, A. et al. (1998) A homeobox gene of the orthodenticle family is involved in antero–posterior patterning of regenerating planarians. Int. J. Dev. Biol. 42, 1153–1158 18 Umesono, Y. et al. (1999) Distinct structural domains in the planarian brain defined by the expression of evolutionarily conserved homeobox genes. Dev. Genes Evol. 209, 31–39 19 Smith, K.M. et al. (1999) CnOtx, a member of the otx gene family, has a role in cell movement in hydra. Dev. Biol. 212, 392–404 20 Sun, H. et al. (1997) Evolution of paired domains: isolation and sequencing of jellyfish and hydra Pax genes related to Pax-5 and Pax-6. Proc. Natl. Acad. Sci. U. S. A. 94, 5156–5161 21 Gauchat, D. et al. (1998) prdl-a, a gene marker for hydra apical differentiation related to triploblastic paired-like head-specific genes. Development 125, 1637–1645 22 Catmull, J. et al. (1998) Pax-6 origins – implications from the structure of two coral Pax genes. Dev. Genes Evol. 208, 352–356 23 Warga, R.M. and Nusslein-Volhard, C. (1999) Origin and development of the zebrafish endoderm. Development 126, 827–838 24 Fu, W. and Noll, M. (1997) The Pax2 homolog sparkling is required for development of cone and pigment cells in the Drosophila eye. Genes Dev. 11, 2066–2078
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25 Wada, H. et al. (1998) Tripartite organization of the ancestral chordate brain and the antiquity of placodes: insights from ascidian Pax-2/5/8, Hox and Otx genes. Development 125, 1113–1122 26 Dahl, E. et al. (1997) Pax genes and organogenesis. BioEssays 19, 755–765 27 Hoshiyama, D. et al. (1998) Sponge pax cDNA related to pax-2/5/8 and ancient gene duplications in the pax family. J. Mol. Evol. 47, 640–648 28 Lundgren, S.E. et al. (1995) Control of neuronal pathway selection by the Drosophila LIM homeodomain gene apterous. Development 121, 1769–1773 29 Han, Z. and Firtel, R.A. (1998) The homeobox-containing gene Wariai regulates anterior–posterior patterning and cell-type homeostasis in Dictyostelium. Development 125, 313–325 30 Wainright, P.O. et al. (1993) Monophyletic origins of the metazoa: an evolutionary link with fungi. Science 260, 340–342 31 Aguinaldo, A.M. et al. (1997) Evidence for a clade of nematodes, arthropods and other moulting animals. Nature 387, 489–493 32 Veuthey, A.L. and Bittar, G. (1998) Phylogenetic relationships of fungi, plantae, and animalia inferred from homologous comparison of ribosomal proteins. J. Mol. Evol. 47, 81–92 33 de Rosa, R. et al. (1999) Hox genes in brachiopods and priapulids and protostome evolution. Nature 399, 772–776 34 Odorico, D.M. and Miller, D.J. (1997) Internal and external relationships of the Cnidaria: implications of primary and predicted secondary structure of the 59-end of the 23S-like rDNA. Proc. R. Soc. London B Biol. Sci. 264, 77–82 35 Ayala, F.J. and Rzhetsky, A. (1998) Origin of the metazoan phyla: molecular clocks confirm paleontological estimates. Proc. Natl. Acad. Sci. U. S. A. 95, 606–611 36 Mokady, O. et al. (1998) Over one-half billion years of head conservation? Expression of an ems class gene in Hydractinia
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symbiolongicarpus (Cnidaria: Hydrozoa). Proc. Natl. Acad. Sci. U. S. A. 95, 3673–3678 37 Schummer, M. et al. (1992) HOM/HOX homeobox genes are present in hydra (Chlorohydra viridissima) and are
differentially expressed during regeneration. EMBO J. 11, 1815–1823 38 Martinez, D.E. et al. (1997) Budhead, a forkhead/HNF3 homologue, is expressed during axis formation and head
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specification in hydra. Dev. Biol. 192, 523–536 39 Technau, U. and Bode, H.R. (1999) HyBra1, a Brachyury homologue, acts during head formation in Hydra. Development 126, 999–1010
Mass spectrometry from genomics to proteomics Large-scale DNA sequencing has stimulated the development of proteomics by providing a sequence infrastructure for protein analysis. Rapid and automated protein identification can be achieved by searching protein and nucleotide sequence databases directly with data generated by mass spectrometry. A highthroughput and large-scale approach to identifying proteins has been the result. These technological changes have advanced protein expression studies and the identification of proteins in complexes, two types of studies that are essential in deciphering the networks of proteins that are involved in biological processes. he elucidation of an organism’s genome is the first and important step towards understanding its biology, and the data created by whole-genome sequencing have significant benefits in fields outside those of genomics and bioinformatics. One area to benefit is that of proteomics. The term proteomics, or more appropriately functional proteomics, describes the ability to apply global (proteomewide or system-wide) experimental approaches to assess protein function. Proteomics has emerged as a new experimental approach in part because mass spectrometry has simplified protein analysis and characterization, and several important and recent innovations have extended the capability of mass spectrometry.
T
Mass spectrometry of biological molecules Mass spectrometers consist of three essential parts (Fig. 1). The first, an ionization source, converts molecules into gas-phase ions. Once ions are created, individual mass-tocharge ratios (m/z; see Box 1) are separated by a second device, a mass analyzer, and transferred to the third, an ion detector. A mass analyzer uses a physical property [e.g. electric or magnetic fields, or time-of-flight (TOF)] to separate ions of a particular m/z value that then strike the ion detector. The magnitude of the current that is produced at the detector as a function of time (i.e. the physical field in the mass analyzer is changed as a function of time) is used to determine the m/z value of the ion. Although mass analyzers are an important (and continually improving) component of mass spectrometers and determine critical performance characteristics, an important innovation for proteomics has been the development of two robust techniques to create ions of large molecules. Matrix-assisted laser desorption ionization (MALDI) creates ions by excitation of molecules that are isolated from the energy of the laser by an energy absorbing matrix. The laser energy strikes the crystalline matrix to cause rapid excitation of the matrix and subsequent ejection of matrix and analyte ions into the gas-phase. Electrospray ionization (ESI) creates ions by application of a potential to a flowing liquid causing the liquid 0168-9525/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(99)01879-X
to charge and subsequently spray. The electrospray creates very small droplets of solvent-containing analyte. Solvent is removed as the droplets enter the mass spectrometer by heat or some other form of energy (e.g. energetic collisions with a gas), and multiply-charged ions are formed in the process. The detection limits that can be achieved with ESI have improved with a reduction in the flow rates1. These ionization techniques have stimulated developments in mass spectrometers to enhance the production of two different types of information. The first type of information is the accurate measurement of molecular weight. To measure molecular weight to the low ppm level, MALDI is used typically in conjunction with TOF mass analyzers. The second type of information, produced by tandem mass spectrometers (MS/MS), is diagnostic of amino acid sequence (Fig. 1b). Many types of MS/MS have been developed2, and new innovations allow greater automation and efficiency in data acquisition. Data can be generated in a data-dependent manner through interaction of the m/z data in each scan with a computer program to control the type of experiment performed3. For example, a scan of the mass range can reveal the presence of several ions above a preset ion-abundance threshold. The computer can signal the instrument to perform tandem mass spectrometry on each of the ions, thus improving the efficiency of data acquisition, particularly during separations when ions appear for only a brief period of time.
Identifying proteins using mass spectrometry data and database searching Mass spectrometers are capable of generating data quickly and thus have a great potential for high-throughput analysis. An essential component to achieving greater throughput is simplifying data analysis. There is a direct relationship between mass spectrometry data and amino acid sequences. Peptide molecular weight measurements are predictive of amino acid composition, and peptide fragmentation information (as described in the glossary) relates to amino acid sequence. Both types of information can be correlated to protein sequences in the database. A single peptide TIG January 2000, volume 16, No. 1
John R. Yates, III jyates@ u.washington.edu Department of Molecular Biotechnology, University of Washington, Seattle, WA 98195-7730, USA. 5