Chapter 15 Mammalian cell biology and proteomics

Chapter 15

Mammalian cell biology and proteomics Jorge S. Burns

The ‘‘hour-glass’’ nature of proteomics dictates that a complex organisation of matter passes through a narrow aperture in order to allow measurements that produce a similarly complex broad data set. The challenge of gathering and interpreting this information so that one could then invert the ‘‘hour-glass’’ data set and fully describe the initial molecular organisation has yet to be met. Nonetheless, just as an hour-glass functions adequately without having every grain of sand return to the exact equivalent of its original location, proteomics has already provided major advances in our understanding of cell biology. Metaphorically, the proteomic shop window might seem like that of a watchmaker, gazed upon by a perplexed cell biologist asking, ‘‘Can there really be so many ways of telling the time? Will self-winding do, or should one go for quartz precision?’’ Hoping to help with informed decisions, this chapter provides an overview of proteomic advances and highlights aspects for practical consideration when investigating human cells, given a very broad number of alternative preparatory methods for determining protein content and function. 15.1

THE PROTEOME IS MUCH MORE COMPLEX THAN THE GENOME

A genome represents the entire complement of genetic material in a chromosome set, with a well-defined, fully sequenceable endpoint. In contrast, the proteome, broadly defined as all the proteins expressed by a cell or organ at a specific time under a specific set of conditions, represents a dynamic and arbitrary endpoint with greater complexity. A practical goal of proteome analysis would be to robustly obtain protein expression data to the same extent now obtainable for mRNA Comprehensive Analytical Chemistry 46 Marko-Varga (Ed) Volume 46 ISSN: 0166-526X DOI: 10.1016/S0166-526X(05)46015-8 r 2005 Elsevier B.V. All rights reserved.

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expression data via DNA microarrays. Although there is a trend towards developing such convenience, it is very unlikely that one simple platform will comprehensively accommodate the broad sample diversity provided by cells, solid tissues and tissue fluids. Ultimately, proteomic and DNA array technologies are complimentary, needing additional studies to determine correlation, structure, subcellular localisation, tissue distribution and function; the foundations for bioinformatic tools [1].

15.2

EXPLORING EXPERIMENTAL CELL MODEL SYSTEMS

The legacy of Edmund B. Wilson (1856–1939), ‘‘The key to every biological problem must finally be sought in the cell, for every living organism is, or a some time has been, a cell’’, makes cell model systems a logical platform for proteomics. Yet their predominance over analysis of tissue samples and biopsies principally reflects a pragmatic choice. One would like to understand disease processes in a fully therapeutic context, however, as described below, such studies are not straightforward. Nonetheless, cell model advantages can compensate for possible limitations regarding clinical relevance. Greater control of the cellular microenvironment not only improves control of experimental design and reproducibility, but also facilitates exploration of methods to enhance the sensitivity and quantification of proteins and their modifications. There are many challenges for the study of proteins in biological systems, including a broad dynamic range of expression, heterogeneity, the complexity of modifications and many technical and preparatory steps prone to the introduction of bias [2]. The binding of chaperones to proteins and biochemical properties such as hydrophobic interactions, non-specific absorption to the surface of affinity matrices or incompatibility with the separation and identification technology can all contribute to a bias. Highly abundant proteins (e.g. serum albumin or cytoskeletal proteins) may have a general non-specific contribution, clouding identification of proteins that may be more specific and relevant. Parameters that may be taken for granted with routine cell culture may nonetheless influence cell behaviour. A recent systematic study on the pH indicator constituent phenol red, showed that doses in the range often used in tissue culture media (5–10 mg/l) increased the response of rat bone marrow cell to anabolic drugs in a fibroblast colony-forming unit assay [3]. The design of proteomic 558

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projects thus requires very careful consideration of how the method might generate artefacts, how these might be monitored and how best to reach an acceptable compromise. For any given study, it will be important to be able to establish reliable reproducibility and efficient technology for the acquisition and analysis of the data. If possible, early independent confirmation of predictable phenotypes and validation strategies will help assess quality control. In many, but not all circumstances, addressing these questions with cell lines can provide solutions that in turn improve analysis of primary tissue and biopsies. From the outset, it is important to ask to what extent is the cell model able to specifically address the question asked? Large-scale proteomic experiments are costly, generate large amounts of timeconsuming data and justify careful planning for anticipation of any logistical problems [4]. For example, when exploring the effects of a growth factor or inhibitor on a cell, it would be advantageous to adopt a time-course-based functional analysis to have internally corroborating data. Changes in protein expression can be correlated to specific temporal changes evoked by activation of the signalling pathway [5]. Although some immortal cell strains may have desirable properties for such studies, they may grow slowly, making comprehensive analysis of low abundance transcription factors and signal regulatory proteins difficult. One may be able to analyse such targets, but the need to reproduce time points and the low molecule per cell ratio, may require cell numbers in the order of 109. Extensive expansion of cells in vitro is itself a contentious issue that may be accompanied by spontaneous genetic and epigenetic changes. Large cell populations are more prone to harbour covert intercellular heterogeneity. Fortunately, the trend for advanced proteomic approaches is to be able to gather information on several thousands of proteins from ever smaller amounts of starting material, typically only 104–105 cells, with even mention of single-cell analysis.

15.3

FINDING FUNCTION WITH SELECTED CELLS; WHEN LESS IS MORE

For proteomic approaches that consist of comparative analysis, closely matched cell model systems that are largely isogenic, make attractive starting points with less variation. Generating such cell systems may take years of careful clonal expansion and analysis as was the case for 559

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breast cancer cell lines that differ with regard to expression of the estrogen receptor [6]. Such a model system and tamoxifen-resistant breast carcinoma xenografts [7] are useful for exploring improvements to the current gold standard therapy [8]. An alternative strategy for generating well-matched controls for drug screening involved taking advantage of cancer cells with defined endogenous alterations of specific genes. Deleting their mutant gene by homologous recombination resulted in a cell line that differed from the parental population by only the single mutant gene. By tagging cell populations with expression vectors for different fluorescent proteins, it was possible to co-culture parental and gene-targeted cells to allow precise internal calibration and control for each assay [9]. An intrinsic advantage to studies that aim to understand a protein’s function by silencing its gene expression is that such approaches mimic physiological gene deletion events. A poignant example, concerns the down-regulated expression of the tumour suppressor protein p27Kip1 in advanced cancers in a number of tissues [10]. Rarely mutated, loss of only one allele can be sufficient for a predisposition to tumorigenesis. This emphasises the importance of the ubiquitin-proteasome pathway that regulates p27Kip1 expression levels [11,12]. Quiescent cells accumulate p27Kip1 without an increase in mRNA or protein synthesis and polymerase chain reaction (PCR)based methods do not necessarily detect its loss in advanced neoplasia. Tools for assisting proteomic profiling of the ubiquitin family of proteases have recently been developed. Protein-based probes targeting the enzyme’s specific active site were successfully applied to complex mixtures of lysates to discover novel ubiquitin specific proteases [13]. System wide analysis of proteases, termed ‘‘degradomics’’ [14] will reveal a hitherto undescribed level of information with important tissuespecific roles and disease involvement [15]. Inactivation of gene expression via RNA interference (RNAi) [16,17] is a rapidly emerging technology likely to play an increasingly important role in proteomic studies [18]. Synthetic small interfering RNA (siRNA) consisting of 21–23 nucleotides of double-stranded RNA can directly interfere with expression of individual genes [19], although in mammalian cells varying degrees of efficiency require empirical testing. Validated libraries are increasingly becoming available and were demonstrably useful in a detailed study combining siRNA and proteome analysis to reveal novel mammalian apoptosis regulators [20]. A ‘‘positive’’ approach for studying a protein’s function would be to test the effects of its overexpression. Many cancer causing oncogenes 560

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are expressed at higher levels in tumour cells than in their normal counterparts but controlling the level of an exogenously expressed gene to ensure equivalent expression is difficult. Often the study is prone to bias because expression from the gene vector’s exogenous promoter may exceed pathological levels. Nonetheless, more elaborate inducible vectors [21] can greatly improve control of gene expression, as can vectors that facilitate careful selection of stable expresser clones [22]. Common to all cell model systems are a number of preparatory steps in the proteomic process before protein identification, including cell lysis, protein extraction and separation. The technical aspects of predominant approaches are described below.

15.4

SAMPLE HANDLING/MISHANDLING AND DATA PROCUREMENT

The proteomic platform is an ambivalent stage for philosophically opposed experimental approaches: (i) a holistic view argues that one should keep the sample for proteomic analysis as intact as possible, versus (ii) a reductionist view arguing that given its complexity, pre-fractionation and intervention for simplification is required. The first approach has greatest relevance for high-resolution separation of human proteins using two-dimensional gel electrophoresis (2-DGE). Notoriously very sensitive to manual dexterity and precision, a series of technological advances have made 2-DGE more user-friendly and better at displaying a broader range of proteins. Ameliorating many variables that compromised reproducibility of carrier ampholyte-based 2-DGE, the introduction of immobilised pH gradient (IPG) 2-D technologies has encouraged wider use of the technology [23]. A key advantage is that it rapidly provides an unforgiving clear ‘‘map-like’’ overview of the quality of the sample while isolating and concentrating thousands of largely intact proteins. Post-translational modifications (PTM) are made obvious by their influence on characteristic spot patterns (e.g. phosphorylations principally alters protein charge, yielding a horizontal chain of spots whereas glycosylations alter both charge and mass to yield diagonal streaks). Retention of Mr and pI information can facilitate detection of spliced protein isoforms when spots with the same identity appear in very different regions of the gel. However, the ability for the map of intact polypeptides to reflect changes in protein expression level, isoforms and post-translational modifications, extends 561

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to artefacts also. IPG technology allows greater amounts of protein to be loaded on the gel than previous ampholyte first-dimension gels, improving the detection of low abundance proteins. Nonetheless, it is still possible to overload a gel, leading to saturation and fusion of abundant protein spots, while low abundant spots fall below the threshold of detection. Of main concern in separating proteins by gel electrophoresis is to what extent reagents might interfere with the migratory behaviour of proteins. A recent review provides a clear overview [24] but some salient aspects are mentioned here. A fundamental parameter that needs to be carefully controlled for reproducible gel electrophoresis is the temperature of the reagents and a consistent ambient temperature in the laboratory. Urea, a commonly used chaotropic reagent for protein solubilisation, can form cyanates at temperatures above 301C that will carbamoylate samples during sample preparation and thereby introduce charge alterations [25,26] but with careful routine procedures the risk of this event is low [26]. Proteolytic enzymes, salts, lipids, nucleic acids, polysaccharides and highly abundant proteins can all influence gel patterns. Protease inhibitors may modify proteins and cause charge artefacts but alternative means of inactivating proteases include precipitation with ice-cold trichloroacetic acid (TCA) with the added benefit that this also removes interfering compounds such as salt. The salt concentration needs to be carefully controlled as it increases the conductivity of the isoelectric focusing (IEF) gel, prolonging the equilibration time. Resolubilisation of the TCA precipitate requires thoroughness, since incomplete precipitation or resolubilisation may introduce inconsistent protein losses. Charged nucleic acids and polysaccharides can interact with carrier ampholytes causing streaky 2-D gel patterns and if abundant, can also obstruct the pores of the polyacrylamide gels interfering with protein migration. The problem of highly abundant proteins is very appreciable when examining blood and plasma, given the relatively high amount of albumin. Although some advocate removal of albumin to prevent it eclipsing results, it is a binding partner for many other serum proteins and its removal is likely to involve variable loss of other protein species. Technological advances that can help improve the dynamic range displayed by 2-DGE electrophoresis include narrow-range pH gradients typically 1–3 pH units wide [27,28]. Six IPG strips overlapping the pH ranges from 3.5–5.0 to 7–10 were able to reveal over three times as many distinct spots than the conventional single IPG strip with a pH range of 3–10 [29]. In addition, large format (93  103 cm) gels can 562

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provide 11,000 spots detectable by autoradiography, with a dynamic range of 105 [30]. With high sensitivity, metabolic radiolabelling, allows spot detection by autoradiography. Straightforward dosimetry and standardised safety procedures in processing samples post-irradiation makes it a readily applicable approach for proteomics. Metabolic radiolabelling can provide quantitative information at every step of the purification and gel loading procedure, plus confirmation that the identified protein was derived from the cell, rather than from undefined components in tissue culture medium (e.g. serum). Although this approach can advantageously provide a dynamic range of five orders of magnitude with visualisation of low abundance proteins, the procedure may not be entirely benign. In human fibroblasts, cell-incorporated 32P o-phosphate elicited a p53-dependent inhibition of DNA synthesis that could bias results, with unequal effects on test and control cells [31]. These concerns extend to the low-energy b-emitter 35 S-methionine, which can globally influence a diverse set of cellular activities [32]. This does not mean that comparative data generated using metabolic radiolabelling is necessarily artefactual, but it should be subject to cautious interpretation and rigorous independent evaluation. Although convenient, traditional Coomassie blue or silver staining does not provide a broad dynamic range for 2-DGE spot quantitation and can reduce recovery of low abundance proteins for MS from the gel. Fluorescent-based methods of gel spot detection have been developed, offering greater dynamic range than the above staining methods. The ability to superimpose corresponding 2-DGE spot positions for comparative analysis was facilitated by labelling the different protein samples to be compared with distinct dyes before mixing them for electrophoresis on the same gel. Use of a standard reference dye label can improve quantitative aspects of 2-D differential gel electrophoresis (DIGE) [33]. The initial situation was not perfect, if overloaded, minimal-labelling dyes could attach multiple dye residues to the proteins leading to unquantifiable streaks in the gel. Even when loaded correctly, with about 20% of molecules of a particular protein covalently modified with one Cy dye molecule, the unlabelled majority may not exactly co-migrate with the labelled protein. Post-staining of gels allowed correct excision for mass spectrometry, but could impair identification. Second-generation saturation-labelling dyes also altered the migration pattern of proteins relative to unstained gels, but the 563

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much improved sensitivity eliminated the need for post-staining preparative gels [34]. An often-criticised limitation of 2-DGE gel technologies is the problem of displaying integral membrane proteins and basic proteins. The popular O’Farrell urea lysis buffer [35] is unfortunately not ideal for solubilisation of hydrophobic membrane proteins. Detergents can prevent hydrophobic domain interactions and prevent protein aggregation and precipitation. Non-ionic detergents (e.g. NP-40 or Triton X-100) are often preferred to anionic detergents such as SDS in order to reduce the risk of introducing horizontal streaks in the 2-D pattern. However, zwitterionic detergents such as CHAPS and SB 3–10 combined with thiourea are more effective at solubilising very hydrophobic proteins [36]. Obvert criticism that 2-DGE technology cannot adequately represent membrane proteins [37] may be deemed too harsh in the light of successful examples [38–42]. Nevertheless, with cell-type and tissue-specific variations, optimising the sample solubalisation buffer for membrane proteins remains an empirical trial-and-error exercise. Broadening analysis, technical progress for 2-DGE analysis in the alkaline pH range has improved visualisation of basic proteins [43,44]. However, a paradox of the 2-DGE approach is how to gather global consistency in light of the wide choice of reducing agents, pre-fractionation procedures, gel chemistry, running conditions and methods of protein visualisation and quantitation [45,46]. Comparison of 2-D gel databases across platforms is not straightforward [47]. Even so, there are excellent examples of quantitative 2-DGE technology providing in vitro data with in vivo relevance to vindicate the 2-DGE approach [48]. At the same time, the growing versatility of mass spectrometry is clearly demonstrable [49]. Beyond serving as a tool to provide identity for proteins from electrophoresis gels [50,51], mass spectrometry coupled with innovative reagents has the potential to be a formidable quantitative explorer of proteomic profiles in its own right. Mass spectrometry (MS) can accommodate a more reductionist approach to proteomics, introducing advantages for quantification and high throughput. Although it is possible to analyse intact proteins [52], mass spectra usually represent the mass to charge ratios of peptides from proteins treated with sequence-specific proteases, most often tryptic peptides. Since certain proteins lack suitably spaced cleavage sites and do not yield tryptic peptides of a suitable size, more comprehensive sequence coverage requires combinatorial use of different proteases. This can circumvent the problem of having to cope with 564

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differences in intact protein solubility, avoiding associated use of detergents that readily ionise and interfere with mass spectra. Technological improvements over recent years have greatly improved sensitivity, mass accuracy and resolution. This has allowed peptide identification to evolve from ‘‘mass fingerprinting’’ (probability-based matching of peptide masses in the spectrum with calculated tryptic peptide masses from protein databases), to more sensitive and specific de novo peptide sequencing methods [53] underscored by more robust validation and statistical procedures [54]. Diligent generation of large data sets identifying hundreds of proteins from the Malaria parasite not only reduced misidentifications but also comparison to the genomic sequence helped resolve ambiguous identities [55]. Similarly, for the human genome, protein identifications will also be enormously helpful for reciprocally proofreading genomic DNA sequences and improving annotation of the genome [56,57]. Given improved physical measurements of proteins and their modifications, the restriction to interpretation of the data is often a computational one [58,59]. Algorithms matching peptide fingerprints to genomic data [60] also extend rules of evidence and help improve confidence in the data set. Traditionally, the spectra acquisition time is much shorter than the relatively long time taken to search DNA genomic databases with MS data and this can be inconvenient when sample is rapidly consumed during analysis. This bottleneck has been addressed by new hardware design, capable of generating exclusion lists so that peptide masses of little interest (e.g., trypsin or keratin peptides) are dynamically eliminated from mass spectra analysis, focusing attention on unidentified and less abundant peptides. A single hardware unit could search the human genome in less than 2 s with faster search times costing 40 times less than an equivalent specification 64 processor cluster [61]. Two principal approaches provided the acclaimed breakthrough for energising the ionisation of peptides without destroying the molecule; namely, electrospray ionisation (ESI) [62] and laser desorption [63] which led to matrix-assisted laser desorption ionization (MALDI) [64]. The MALDI ion source was traditionally coupled to time-of-flight (TOF) mass analysers, whereas ESI was usually coupled to iontrap or triple–quadrapole tandem MS instruments. The latter, also referred to as MS/MS, has two consecutive MS stages, in the first MS step a peptide ion is isolated and then collisions with an inert gas provided appropriate energy for breaking the amide bonds of this precursor ion into product ions that are sources for the second MS step. Fragmentation of 565

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non-amide bonds can complicate the spectrum, but principally, it consists of truncated versions of the precursor ion, classified as b-ions (charge retained by the N-terminal) and y-ions (charge retained by the C-terminal). By trapping these product ions, they can be fragmented further (MSn) providing in-depth characterisation via linear ion trap instruments [65]. Hybrid instruments, with option for interchangeable MALDI and electrospray sources, can accommodate the convenient reanalysis of samples advantageous to the MALDI source workflow. The Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FT-ICR), introduced in 2003, provides an improved linear ion trap storing the ions in a cylindrical field that has a much higher capacity. The peptide ions orbit within a strong magnetic field at a precise frequency that is inversely proportional to their mass to charge ratio. FT-ICR detector measurement of frequency can provide much higher mass accuracy and sensitivity, enabling analysis of very complex mixtures of thousands of peptides with greater speed. The improved quality spectra reduced ambiguity when searching genomic databases, increasing confidence in protein magnidentifications by two orders of magnitude [66]. The implications for the biologist are that MS instrumentation has evolved from mass analysis and database matching to de novo peptide sequencing, providing more specific and sensitive identification and structural information. How does one simplify complex cell or tissue protein mixtures to improve resolution from the mass spectrometer [67,68]? There are a wide number of alternatives to 2-DGE separation protocols, including a combination of one-dimensional SDS-PAGE and high-performance liquid chromatography (HPLC). Gel lanes excised into slices ranging known molecular weights can provide fractions for independent analysis runs, thereby improving the resolution by HPLC and MS. This method generated a large data set of 2341 non-redundant human proteins from immortalised cell lines [69]. An alternative, post-digest approach, separates the mixture of peptides in two dimensions using a strong cation exchange (SCX) column to separate the peptides on the basis of charge, combined with a reverse-phase (RP) column to separate the peptides on the basis of hydrophobicity. Packing the SCX and RP columns at opposing ends of a single capillary column, minimised sample loss between the two separation dimensions and the entire system was coupled directly with MS. This online, multidimensional protein identification technology (MudPIT) [70] successfully identified 160 candidate midbody proteins important in the critical phase of cell division termed cytokinesis [18]. A recent comparison of immobilised 566

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pH gradient gels versus SCX chromatography in the first dimension, suggested that the IPG strip-based method could provide better peptide separation with 13% more protein identifications [71]. The binding of chaperones to proteins and biochemical properties such as hydrophobic interactions, non-specific absorption to the surface of affinity matrices or incompatibility with the separation and identification technology can all contribute to internal bias. Highly abundant proteins (e.g. serum albumin or cytoskeletal proteins) may have a general non-specific contribution, clouding identification of proteins that may be more specific and relevant to a particular experiment. Reviews of different strategies for peptide separation [67,72] concede that none are perfect, but as the separation methods become increasingly dedicated towards proteomic objectives their performance will continue to improve.

15.5

CONVENIENT COMPARTMENTS, MINING ORGANELLES

It is very logical to exploit compartmentalised cellular organisation in order to reduce sample complexity prior to analysis and explore subcellular proteomes [73]. The two major steps consist of disruption of the cellular organisation and fractionation of the homogenate to separate the different organelles [74–76]. The divergent protein properties of different organelles can be used to increase the visualisation of low abundance proteins. A convenient reproducible stepwise extraction method has been validated and commercialised [77] and though it may not match the purity of more specialised organelle extraction protocols it can provide a rapid and helpful selective enrichment of four subcellular fractions and confirm redistribution of proteins in response to signalling molecules, e.g. translocation of phosphorylated mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-KB) from the cytosol to the nucleus upon cellular stimulation with tumour necrosis factor a (TNF-a). The plasma membrane has received much attention; it is host to about a quarter of all cellular proteins, and most drugs target proteins on cell surfaces. However, homogenisation of a pure plasma membrane (only 2–5% of the total membrane component of mammalian cells) is made difficult by the co-distribution of proteins from membranes in other cell organelles, such as mitochondria (35–60%) or endoplasmic 567

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reticulum (ER) (20–40%). Several alternative procedures for cell disruption and subsequent enrichment have been described. Different samples have different optimal procedures, e.g. after cell disruption and homogenization, cultured cells may be more difficult to fractionate than most tissues, possibly reflecting differences in cytoskeletal organisation. A simplified approach reporting high plasma membrane purity, involved attaching viable cells to nitrocellulose-treated DEAESephadex beads and subsequently shearing the cells by hypotonic lysis, agitation and sonication. The beads served as plasma membrane carriers; ultimately, marker enzyme activities suggested a 12–16-fold enrichment of plasma membrane proteins with contamination from internal membrane markers decreasing through isolation steps to less than 1% [78]. Comparing methods of cell disruption, Watarai et al. [79] compared nitrogen cavitation versus dounce homogenisation. Highpressure nitrogen cavitation was an effective method for obtaining a complete cell lysis allowing subsequent isolation of subcellular fractions of yield and purity greater than mechanical homogenisation. However, optimal nitrogen cavitation conditions need careful adjustment for different cell types and tissues. Frozen samples, e.g. small biopsies, present a specific situation whereby broken nuclei and release of DNA can cause aggregation of organelles, complicating their isolation. Recent alternative high-speed shearing-based methods with digestion of proteins on non-solubilised membranes have largely circumvented these problems, providing one of the most extensive analyses of murine brain membrane proteomes to date [73]. At high pH, sealed membranes are disrupted without denaturing the lipid bilayer or releasing integral proteins. Moreover, high pH attenuates the activity of proteinase K, so that it cleaves proteins to form peptides (6- to 20-mers) that are optimal for liquid chromatography and MS. These qualities formed the basis of a method that allowed rapid comprehensive characterisation of membrane proteins, including information regarding their topological orientation within the bilayer [80]. Often, a particular cell type needs to be sorted before analysis and there has been much progress in the field of high-speed cell sorting [81]. However, plasma membrane analysis is inherently vulnerable and sensitive to environmental changes. The cell separation method can play an integral role in modifying plasma membrane quality before analysis. Although fluorescent activated cell sorting (FACS) or magnetic cell separation (MACS) did not affect membrane viscosity, 568

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hydrodynamic forces imparted by both methods were able to modify the plasma membrane in a cell type-dependent manner [82]. For certain cell types, gentler immuno-panning-based methods for isolating rare cell subtypes from biological fluids may be advantageous [83]. The plasma membrane is far from being a homogeneous lipid bilayer; cholesterol-rich lipid microenvironments on the cell surface, known as lipid rafts, have an important role in signal transduction, by providing a structure that favours protein–protein interactions [84]. To gain greater specificity for proteome analysis of proteins localised to lipid rafts, Foster et al. [85] devised an ingenious comparison between two cell populations, one of them treated with a cholesterol-disrupting drug to break up lipid rafts. A quantitative approach using stable isotope-labelling with amino acids in cell culture (SILAC) (see Section 15.7 discussed below), allowed an unbiased subtractive comparison between cells with intact rafts versus cells without rafts. Two raft isolation methods and different cholesterol disrupting drugs were used to derive a comprehensive data set. The degree of enrichment allowed the identified proteins to be categorised as belonging to either total membrane fractions, detergent-resistant membranes or lipid rafts. A key advantage of this approach is that one-third of the proteins could be distinguished as non-specific copurifying proteins and it allowed a comparison of the raft isolation methods, detergent-resistance being preferred. The data revealed 241 authentic raft proteins with a significant number of tyrosine and serine/threonine kinases and phosphatases, together with heterotrimeric G protein subunits to support a role for lipid rafts in signal transduction and coordination. In contrast to hard-to-purify plasma membranes, successfully specific methods for isolating mitochondria have facilitated acquisition of high-quality proteomic data for this organelle. The numerous experiments used to determine the mitochondrial proteome in different species can serve as a useful benchmark for determining the sensitivity and specificity of the proteomic approach [86]; values of 50–60% for these parameters are not uncommon. One of the most comprehensive studies in mammalian cells compared the mitochondria from murine heart, brain, liver and kidney [87]. This revealed tissue-specific differences in mitochondrial composition, corroborated by concordant tissuespecific differences in mRNA abundance for the identified proteins. Furthermore, integrating proteomic and genomic analysis revealed a pattern of 643 co-regulated genes, implicated in mitochondrial biogenesis and function. 569

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The organelle with the most extensive data for a cellular compartment is the nucleosome. This serves the critical function as the site of ribosomal gene expression and ribosome assembly. Largely the result of a prolonged collaboration between two laboratories, the latest study has explored the kinetics with which protein components enter and leave the nucleolus [88]. The flux of 489 endogenous nuclear proteins in response to metabolic inhibitors was consistent with evidence that the nucleolar proteome changes significantly in response to growth conditions. Increased sensitivity, resolution and peptide sequencing speed provided by an FT-ICR instrument was evidenced by proteins not found in earlier studies [89]. Proteomic profiling revealed a broad range of proteins in the nucleosome, including 126 novel uncharacterised proteins. Kinetic studies following inhibition of transcription by actinomycin D treatment consistently showed accumulation of at least 11 proteins, illustrating modular characteristics. Different factors had different redistribution patterns and kinetics, with clues to potential functional complexes emerging from coordinated behaviour, e.g. subunits of RNA polymerase I. Accumulation of certain proteins in the nucleolus in the absence of ongoing rRNA synthesis challenged a simplistic definition of the nucleosome as a ribosome factory. Large-scale profiling will lead to an understanding of additional functions coordinated with environmental growth conditions. Proteomic analysis can provide results that lead to the discovery of new structures. One clear example observed in cell culture was termed spreading initiation centres (SIC). These were found to contain focal adhesion proteins that formed transient structures surrounded by an actin sheath, containing RNA-binding proteins. The unexpected finding of a ribonucleoprotein complex in this context was functionally corroborated by an increase in cell spreading when antibodies to RNAbinding proteins were applied to the cells [90]. Proteomic analysis was also responsible for a paradigm shift in our understanding of the phagosome. A key aspect of dead cell removal and a defence against infection, the phagocytic mechanism of macrophages allows these specialised cells to engulf particles as large as themselves, encapsulating them in a membrane-bound organelle known as the phagosome. Such particle internalisation is made possible by the rapid recruitment of membrane from internal pools. Exploring the mechanism involved in phagolysosome biogenesis by identifying protein components using 2-DGE, [91] showed that the ER rather than plasma 570

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membrane, served as the principal source of membrane for phagosome formation in macrophages.

15.6

ZOOMING IN ON MODIFICATIONS

Truly extending analysis beyond what is achievable with gene expression studies, proteomics has unique potential for deciphering how cells communicate between and within themselves. With over 200 different types of PTM, proteomic analysis has had to prioritise aims in line with technological developments [92]. The most explored PTM has led to the coining of new names, ‘‘phosphoproteome’’ and ‘‘kinome’’. Reversible phosphorylation by protein kinases serves as a mechanism to influence protein activity in virtually all signalling pathways [93,94]. Defining the sites of protein phosphorylation and their status are challenging tasks. These are transient, reversible modifications, of different stability (phosphotyrosine is more stable than phosphothreonine or phosphoserine). Furthermore, out of an in vivo context, kinase specificity is often less stringently controlled, leading to false-positives. An early limitation of MS was that although the peptides analysed provided a high degree of confidence with regard to identity, incomplete sequence coverage did not allow the mapping of all PTM. Greatly improved MS based methods have evolved rapidly; nonetheless, 2-DGE may be considered advantageous with regard to PTM, since they are readily displayed with altered protein migration. In an early example, specific identification of phosphoproteins was routinely possible with careful sample preparation; however precise identification of the phosphopeptide required more protein than was typically obtainable from IEF gels [95]. Combining in-gel digestion with nanoscale immobilised Fe(III) affinity chromatography (IMAC) columns for capturing and enriching phosphoproteins enhanced the sensitivity and improved the detection of phosphopeptides [96]. Modern approaches to staining gels specifically for phosphoproteins with fluorescent dyes can be faster and more convenient than relying on autoradiography after 32P or 33P [97]. Generally, such methods can be useful for providing an initial overview, but for greater resolution and faster throughput, MS has been coupled to other enrichment and purification strategies. Without the sample simplification and resolving power of 2-DGE, alternative strategies needed to address the problem of complex mixtures and cope with competition from abundant proteins, including unphosphorylated 571

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forms of the same peptide. Moreover, phosphopeptides larger than 2500 Da are difficult to sequence by MS/MS because they often fail to ionise efficiently. Fortunately, these challenges have been addressed with more sophisticated protein chemistry. For example, chemical modification of peptides before the use of IMAC column can prevent non-specific binding of non-phosphorylated peptides containing acidic residues [98]. Chemical modification can be used more directly in the identification, by replacing the phosphate group with an affinity tag that is more compatible for MS [99]. Although the latter method was not perfect (a 2% probability side reaction could introduce the affinity tag into some non-phosphorylated peptides) it was very capable of enriching phosphopeptides from complex mixtures. A particularly apt chemical modification method, involved converting the phosphoserine and phosphothreonine residue into a proteolytic target site. Thus, phosphorylated sites became C-terminal peptide residues, facilitating interpretation of the MS/MS spectra [100]. Phosphatase inhibitors can help provide a ‘‘smoking gun’’ trail of phosphorylated proteins involved in signalling pathways. Although information about how events might be coordinated is compromised, it allows detection of otherwise transient short-lived phosphorylation events. Calyculin A, a serine/threonine phosphatase inhibitor induced high levels of protein phosphorylation in murine B cells allowing Shu et al. [101] to identify 107 phosphorylated proteins and 193 phosphorylation sites. Only 42 of these proteins were previously known to be phosphorylated, so the study readily provided many new B-cell phosphoprotein candidates. However, the use of a phosphatase inhibitor is likely to cause indirect perturbation of signalling pathways, so the exact relevance of the phosphorylated protein identified needs to be corroborated independently. A wide range of methods can be used to complement MS based phosphoproteomic approaches [102]. One means of independently identifying the kinases targeting a particular phosphorylation site exploited the equilibrium nature of phosphorylation reactions [103]. In the presence of high concentrations of ADP in vitro, protein kinases effectively act as slow protein phosphatases. Incubating the phosphorylated form of the substrate of interest with a variety of kinases could test which is most capable of dephosphorylation, implicating it as the most likely physiologically relevant kinase. A source of complexity when using MS/MS for characterisation of PTM, stems from the fact that the collisionally activated dissociation 572

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used to generate multiply charged peptide ions can also energise the loss of phosphates from amino acid residues (H3PO4 for Ser, Thr or HPO3 for Tyr). With more gentle energy properties, ion fragmentation by electron capture dissociation (ECD) produced complementary spectra that improved preservation of PTMs, generating more sequenceinformative backbone fragments with negligible loss of phosphate [104]. Others have taken advantage of the fact that phosphorylated tyrosine residues produce the characteristic HPO3 fragment ion under defined conditions. Monitoring for the presence of this ion in a precursor ion experiment using a quadrapole time-of-flight mass spectrometer can reveal the presence of phosphorylation on tyrosine while simultaneously localising the tyrosine-phosphorylated peptide by direct sequencing. This phosphotyrosine-specific immonium ion scanning (PSI) approach [105] was certainly helpful, given that phosphorylation on tyrosine is relatively rare in comparison to phosphorylation of serine and threonine. Broad-scale analysis of phosphorylation has reached a global kinetic perspective [102]. Illustrating the versatility of the SILAC method, Blagoev et al. [106] were able to detail the activation profiles of different categories of epidermal growth factor receptor (EGFR) effectors. Using enrichment with phosphotyrosine specific antibodies and quantitative approaches that will be described below, a time course analysis of the first 20 min after stimulation with EGF ligand identified 81 signalling proteins and 31 novel effectors, implicated by virtue of their phosphotyrosine status during stimulation. Since the method enriched phosphopeptides instead of phosphoproteins the temporal order of events in a signalling pathway could be resolved for individual phosphorylation sites. Ultimately, a comprehensive protein phosphorylation analysis benefits from a combinatorial approach, blending the merits of different methods.

15.7

QUANTITATIVE MASS SPECTROMETRY

In broad terms, proteomics has included open-ended discovery-based research and more targeted hypothesis-driven research. Growing convergence reflects improved databases and relevant hardware with new types of MS that enable higher throughput with improved mass accuracy. Discovery research initially implied ‘‘mapping’’ the proteins present in a semi-quantitative manner. However, emphasis on finding 573

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key determinants of different cellular states required a much more quantitative approach, to focus on a subset of differences among otherwise largely unchanged protein profiles. As can be appreciated from the above examples, cellular protein characteristics that can facilitate biological analysis include specific localisation within the compartments of functional organelles, defined mechanisms of PTM, specific interacting motifs such as –SH2 and –SH3 domains [107], known mechanisms for protein degradation such as ubiquitination and the specificity of enzymes and antibodies for their substrates and antigens. The dynamic range of proteins in a cell population or tissue exceeds the dynamic range measurable by proteomic instruments so an accommodating strategy is to specifically enrich or fractionate the sample. This simplifies the complexity of the sample while enriching the desired proteins. Additional steps before MS analysis may introduce artefacts and bias, but approaches that can retain quantitative information and minimise these problems are emerging. Sensitive high throughput methods for the quantitative analysis of changes in protein expression are converging on the principle of having a comparative analysis with the proteins in one sample enriched in stable heavy isotopes [108]. Acronyms include (i) isotope coded affinity tag (ICAT) that targets cysteine containing peptides through reactive sulphydryl groups, reducing the complexity of the sample [109]; (ii) stable isotope labelling in culture (SILAC) that relies on metabolic labelling [110]; (iii) enzymatic labelling, deuterium exchange mass spectroscopy (DXMS) using heavy [18O] water and trypsin, requiring a high-resolution mass spectrometer for complex samples [111]; (iv) absolute quantification (AQUA) relying on a synthetic internal standard peptide, introduced at a known concentration during cell lysate digestion as a reference; and (vi) isotope-coded protein label (ICPL), based on stable isotope tagging of the frequent free amino groups of isolated intact proteins [112]. A key distinguishing feature between the above is whether the heavy isotope label is introduced as a metabolite, or chemically when the proteins are being processed for MS. Without the need for metabolic labelling, ICAT can more readily be used for the analysis of tissues. However, the protocol is relatively expensive and it is restricted to proteins containing cysteine residues (though this may be seen as advantageously simplifying very complex samples). The original cysteinereactive reagent contained a biotin tag and a linker with either eight or no deuterium atoms that remained attached to the peptide. This 574

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influenced elution times in liquid chromatography and was susceptible to unpredictable fragmentation that further complicated interpretation of the MS/MS spectrum. Therefore, modified ICAT reagents for improved liquid chromatography co-elution [113] and cleavable biotin moieties have been derived [114] as well as alternative cleavable affinity tags [115]. Using a solid-phase version of ICAT to covalently capture cysteinyl peptides allowed the isolation of cysteine-containing peptides and the stable incorporation of isotopes to be combined in one step, helping to improve simplicity, efficiency and sensitivity [116]. Notably, though the solid-phase method captured peptides after digestion, the original ICAT method labelled proteins before proteolysis and would thus be more appropriate for gel electrophoresis separation of proteins. A recent re-evaluation of ICAT-MS versus gel-based strategies, acknowledged high quantitative reproducibility with both techniques, but ICAT failed to show superiority over 2-DGE with regard to bias for acidic proteins and under-representation of small proteins less than 10 kDa and hydrophobic proteins [117]. Protocol modifications, such as use of the endoproteinase Lys-C to help generate more basic peptide fragments might ameliorate some MS shortcomings. The SILAC metabolic labelling method is versatile, allowing different isotopes to specifically label different cellular states that can then all be analysed simultaneously [118]. Recent studies used three isotopic variants of arginine; normal 12C614N4 arginine, 13 C614N4 arginine and 13C615N4 arginine have explored hard to quantify scenarios mentioned above, such as the rapidly changing phosphorylation status of phosphotyrosine proteins and the flux of proteins within an organelle. Cells were cultured specifically in one of each of the three types of arginine for sufficient doubling to ensure saturated uniform labelling of the arginine-containing proteins. The major quantitative advantage of SILAC is that from the earliest stages of harvest, cells can be pooled and processed in parallel, thereby equilibrating any procedural bias. The arginine-containing peptides can be traced to their corresponding sample on the basis of the telltale isotope label mass differences of 6 and 10 Da, causing a consistent horizontal peak shift in the proteomic spectrum. The height of the peak is proportional to the relative abundance of the peptide, thus ratios between samples can be calculated, with the set of peptides constituting each protein providing corroborative ratios. Tailored software algorithms automate the process of selecting the ‘‘peak family’’ corresponding to each peptide. 575

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It should not be overlooked that the essential amino acids commonly used for metabolic labelling, arginine and lysine, are biologically active dietary compounds that significantly influence cellular phenotypes [119,120]. Reagent quality and the concentrations used need to be carefully controlled. Arginine and lysine residues are themselves the targets of a stable PTM, methylation, which increases their hydrophobicity. Use of heavy [13CD3]S-adenosyl methionine directly labelled the PTM, thereby greatly enhancing and simplifying its detection [121]. Among the novel findings were three previously unknown methylation sites for the Ewing’s Sarcoma (EWS) protein. This study benefited from high mass accuracy to distinguish between acetylation and trimethylation of lysine residue, which differ in monoisotopic mass by just 0.03639 Da. High mass accuracy and high mass resolution coupled to accurate retention times may suffice for quantitative proteomics [122]. Reproducible chromatographic separations and new software algorithms formed a configuration that, given careful sample preparation, could compare differentially expressed proteins from component information; retention time, mass and signal response [123]. The maximal duty cycle of the Q-TOF mass spectrometer provided a rich spectrum data set that sufficed for systematic analysis of the detected peptide components referred to as accurate-mass retention time (AMRT) components. This strategy used Expression Informatics software able to automatically adjust the ion detection threshold over time as a function of the dynamic range within the MS data. This was analogous to the Dynamic Range Enhancement Applied to Mass Spectrometry (DREAMS) approach that involved data-directed injection of the most abundant ions before ion accumulation in the ion cyclotron resistance trap. Elimination of the major ions in this way prevented them from crowding the trap and allowed selective accumulation of lower abundance species for a greater period of time, thereby significantly improving sensitivity with extension of the dynamic range [124]. Such sophisticated high throughput ultrasensitive analysis is largely confined to specialist laboratories with a detailed understanding of instrument calibration and necessary proprietary modifications [125]. The proofs of principle obtained so far indicate that in combination with nanoscale sample processing techniques [126], one may look forward to improved characterization of low abundance proteins and small clinical samples.

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15.8

THE CHALLENGE OF CLINICAL PROTEOMICS

Cancer provides one example of a very complex and intensively studied pathological situation likely to benefit from the application of proteomics. An intrinsic complication is the associated evolution of cell and tissue heterogeneity within the tumour. Although the hallmark of a proliferative phenotype has provided many cancer cell lines, the most significant criticism related to experiments in vitro is that the cell lines often represent atypical examples with only partial relevance to the clinically presented early tumour. Moreover, without sophisticated tissue-engineering methods, the cell culture environment only poorly mimics the situation in vivo. Proteomic methods are increasingly being aimed at direct analysis of clinical samples [127]. Among the most sought information about a molecule’s association with disease is its prognostic significance. The paraffin-embedded biopsy provides well-preserved tissue macromolecules for retrospective analysis long after patient outcome has been determined. Formalinfixed tissues retain excellent histomorphology, however, random crosslinking of proteins during fixation preclude proteomic analysis. Ethanol-fixed, paraffin-embedded tissues provide a more tractable resource for proteomic analysis [128]. The diagnostic pathologist is often guided by changes in the tissue architecture seen in stained histopathological sections, but we are still at the early stages of translating how different molecular events contribute to such changes. One of the first experimentally tested examples in human neoplasia, associating molecular events to corresponding altered tissue architecture, involved thyroid tumorigenesis [129,130]. The ras oncogene could initiate events leading to a histology typical of ‘‘follicular’’ tumours and the ret oncogene could initiate ‘‘papillary’’ tumours. These observations were consistent with the previously described incidence of mutations in these genes in the relevant subclasses of thyroid tumour. Imaging MS represents a proteomic technology that may directly address such relationships [131]. Tissue samples are used to derive contact blots on a membrane target or as sources for direct analysis via laser capture microdissection [132]. MALDI MS lends itself to the analysis of such samples, since the frozen thin tissue section can be mounted on a stainless-steel or conductively coated glass target plate, then carefully coated with a solution of energy absorbing matrix and dried before introduction to the vacuum inlet of the mass

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spectrometer. Precisely controlled consecutive laser spots can then be fired to generate a mass spectrum from molecules within the irradiated area. Typically, this generates signals for 500 – 1000 individual proteins in the molecular weight range 2000 to over 20,000 kDa [133,134]. The advantage of MALDI MS to preserve information concerning the localisation of molecules in a sample allows high-throughput systematic analysis of each tissue section for protein-specific maps correlated with the tissue architecture. Furthermore, this approach can be used to monitor the tissue distribution of drugs and metabolites [135]. In general, the most intense signals come from the most abundant proteins, but exact quantitation and sensitivity is hard to determine. Nonetheless, a given tissue type can generate specific protein profiles that are highly reproducible for a set of serial sections. Early reports showing that this [136] and alternative approaches that generate characteristic protein patterns may help with the subclassification of solid tumours [137] is encouraging, but there is reason to be guardedly prudent when using proteomic patterns for diagnosis [138,139]. A successful means of selecting cell clusters from stained tissue sections to overcome heterogeneity and obtain cell type-specific analysis involves laser microdissection. This has been combined with MS techniques, e.g. surface-enhanced laser desorption ionization/mass spectrometry (SELDI/MS) to generate reproducible MS spectra from 500 to 2000 cells [140]. Proteome analysis of tissues should ideally take into account the complex interactions between the cells and their microenvironment. For example, the extra-cellular matrix (ECM) consisting of polymerised collagens, elastin, structural glycoproteins, adhesive laminin, glycosoaminoglycans and fibronectin, forms a dynamically arranged mesh within the fluid of the interstitial space. Far from being an inert scaffold, the ECM is critical for regulating cell behaviour via cooperative signalling between ECM fibre proteins, the cytoskeleton of the cell cytoplasm and the protein–chromatin nuclear matrix [141]. This in turn influences the co-localisation of transcription factors and influences tissue-specific gene expression. Furthermore, when studying tumours, specific microenvironmental anomalies can be related to inefficient vascular function, most often characterised by hypoxia and acidic pH values [142]. As an adaptive response to deprivation of oxygen and nutrients, hypoxia-inducible factor 1 alpha (HIF-1a), regulated at the post-translational level, stimulates the transcription of several genes ultimately associated with the induction of glycolysis and 578

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angiogenesis [143] as well as stabilising the wild-type p53 tumour suppressor protein [144]. Metabolic alterations in these stressed microenvironments can also include altered expression of hexose transporter proteins [145] which have a polarised distribution in cultured cells [146]. The polarised non-random distribution of proteins in cells is yet another parameter that should ideally be preserved if proteome analysis can expect to describe in vivo events. Immediate cryopreservation of the biopsy specimen might go a long way to preserve cellular phenotypes, but this option is not always possible. Thus when concerned with human proteomic projects, careful biopsy management and collaboration from all staff involved including surgeon and pathologist is key to maintaining sample quality. Once removed from the patient and processed by the pathologist a tissue will have undergone a significant number of inevitable changes, but with careful management it is usually possible to place the tissue within a short space of time under culture conditions that maintain high cell viability. Despite limitations, improved methods are emerging for preservation of the tissue in a state that most closely reflects its state in vivo. Early phases of tissue culture will involve a dynamic acclimatisation period, followed by acquisition of an equilibrated, relatively steady state that allows more reproducible proteomic measurements. A number of studies employing the NASA rotating wall vessel (RWV) bioreactor collectively agree that this method of cell culture permits the formation and maintenance of fundamental characteristics present in tissue structures, thereby improving upon conventional monolayer cell culture conditions [147]. This clinostat retains the relative positions between free-floating cells and their substrates, co-localising particles that can have different sedimentation rates, providing high mass transfer rates and oxygenation without turbulence and extremely low shear forces (approximately 0.5 dyn/cm2) [148], considerably less than those generated in stirring vessels but more than found in static cultures. Tumour cell lines can grow as three-dimensional spheroids that rotate as a solid body with evidence of intercellular interactions and the development of cell-type-specific architectures [149]. Evidence of correct cellular organisation of the dispersed cells included the formation of apical brush borders, polarised epithelial cells and deposition of an extracellular matrix and basal lamina. Notably, a tissue culture medium with a more physiological blend of glucose, galactose and fructose [150] was used. Protein and gene expression studies have shown that rotating wall vessel culture induced changes in a subset of genes [151]. There was improved 579

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maintenance of long-term cultures of functional hepatocytes [152] and positive indications that this method of culture improved the secretory function while reducing the potential immunogenecity of transplantable tissues [153]. Blood constitutes one of the most readily obtainable tissues for diagnosis. Biofluids such as human serum and blood plasma do not lend themselves to interpretable genetic microarray analysis, justifying the priority given by the international Human Proteome Organization (HUPO) to characterise plasma and serum with the Plasma Proteome Project (PPP) [154]. High throughput diagnostic studies have made use of SELDI protein chip technology to selectively enrich for specific subclasses of proteins with specific chromatographic resins [155]. Specialised algorithms analysed the MS spectrum to define a proteomic pattern or ‘‘fingerprint’’ characteristic of diseased states. The limitations implicit in use of a diagnostic pattern rather than directly identifying the proteins that generate it are increasingly being addressed by coupling the SELDI technology to high mass accuracy spectrometers for protein identification [156]. Nonetheless, sample fractionation via capillary electrophoresis can generate richer data sets [157] and this approach forms the basis of most MS/MS biomarker discovery strategies that aim to identify diagnostic proteins from the outset. Proteomic analysis of biofluids [158] has enhanced awareness that shed membrane microparticles or exosomes, carrying cytoplasmic components of the original cell, may play a significant role in mediating longrange signalling within the bloodstream, influencing vascular function [159]. Exospores isolated by differential centrifugation from urine expressed proteins known to be involved in renal and systemic diseases [160], suggesting this may be a convenient and efficient route for biomarker discovery in urine. Few studies have completed the golden circle of obtaining data unique to the proteomic approach with discovery of a protein target verified by evidence for therapeutic significance. The elegant study of Oh et al. [161] addressed the important issue of drug accessibility by comprehensively mapping tissue-induced endothelial cell surface proteins in vivo. This was achieved by silica-coating the luminal endothelial plasma membranes before subcellular fractionation of whole tissues. 2-DGE high-resolution protein maps of the major organs confirmed the powerful 20-fold enrichment of the endothelial plasma membrane isolation method adopted, revealing characteristic signatures for endothelia from different tissues. Subtraction analysis and 580

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bioinformatic confirmation of structure, glycosylation and membrane orientation, led to a shortlist of 11 proteins with extracellular domains. Western blot analysis confirmed the MS identifications and also highlighted that two proteins were only found in endothelial plasma membranes from lung. One of these proteins APP, specific for normal lung tissue, was absent in lung tumour vasculature. In contrast, Annexin A1 (Ann A1) was only found in tumour endothelial plasma membrane and a single dose of 100 mCi 125I-AnnA1 antibody extended survival of tumour bearing rats in 90% of cases. Human tissue sections of solid tumours showed Anna1 was selectively expressed on the neovascular endothelium of multiple solid tumours, suggesting that this may be a target helpful for treating human disease.

15.9

PROTEOME ANALYSIS OF STEM CELLS; BACK TO THE FUTURE

Stem cells, endowed with ability for self-renewal and also generation of daughter cells with multipotential differentiation capacity, epitomise Edmund B. Wilson’s quotation. Extensive interest in their characterisation has emerged from their demonstrable value as therapeutic agents [162]. However, the stem cell is elusive and tends to be different in each instance, making it a particularly challenging subject for interpretation of proteomic data. Indeed, it has been argued that perhaps ‘‘stemness’’ relates more to a transient cell state than an inherent characteristic of a particular cell type that might be defined by a genetic or proteomic signature [163]. Proteome analysis will be advantageous for studying contextdependent stem cell biology, especially with regard to molecules and signalling mechanisms regulated at the post-translational level. Fetal Antigen 1 (FA1), a soluble product of the gene Dlk1 (delta-like 1), provides a good example. Pseudonyms from polymorphic variants include Pref-1 (preadipocyte factor 1), pG2, SCP-1 and ZOG. This poorly understood member of the Notch epidermal growth factor (EGF)-like family of ligands and receptors is thought to be involved in cell fate decisions and differentiation. Widely expressed in embryonic tissues, it has a more restricted expression pattern in adults, including presumed endogenous stem cells in regenerating liver [164]. Although it lacks a typical interaction domain, Dlk1 is considered to interact specifically with Notch1 [165]. Cell–cell signalling via Notch receptors involves 581

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PTM and ligand interactions that regulate proteolytic events for release of the Notch intracellular domain (NICD) from the plasma membrane. Translocated to the nucleus, NICD displaces a co-repressor to form part of a transcriptional activation complex [166]. Dlk1/Pref-1 has been found to be expressed in bone marrow derived human mesenchymal stem cells (hMSC) and its overexpression in these cells can inhibit their differentiation to mature osteoblasts and adipocytes [167]. The decisive response of any stem cell to extracellular stimuli, selfrenewal versus commitment to a differentiation pathway, has been studied with analysis of the phosphorylation status of 31 intracellular signalling proteins over three time points in the context of 16 microenviromental conditions that combined cyotokine and extracellular matrix components [168]. Matrix components could clearly synergise with cytokines. Murine ES self-renewal signals, e.g. leukemia inhibitory factor (LIF) were notably context dependent. An important general conclusion from the study was that in the multivariate analysis, tested components might have only a small effect, contrasting with the more dramatic effect from single molecules when experimental conditions are constructed to sharply feature differences (e.g. by using inhibitors). The practise of arbitrarily selecting ‘‘significance’’ thresholds of two-or threefold needs to be reconsidered for interpretation of results with a systems biology perspective. The latter example involved analysis of pre-selected signalling molecules, an approach characteristic of protein microarrays [169] that will greatly facilitate high-throughput analysis. The design of microarrays will evolve in the wake of open-ended exploration with more conventional approaches. Quantitative proteomic methodology, such as SILAC, has allowed comprehensive analysis of dynamic signalling pathways governing osteoblastic differentiation and bone formation [170] with relevance for novel therapies to counteract osteoporosis. Multipotential and pluripotential stem cells are sensitive responders to the microenvironmental signals elaborated by tissue engineering approaches [171]. Compared to static cultures, collagen-embedded rat neural stem cells showed improved differentiation in the RWV bioreactor [172] and similarly there was improved generation and viability of human embryoid bodies derived from embryonic stem cells [173]. Future applications of proteomics will hopefully continue to provide increasingly dynamic information, revealing how molecular interactions are regulated and coordinated in signalling pathways that are critically important to disease processes. Advances that will contribute 582

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to this are not confined to just proteomic platforms per se, but include complementary technologies. Microarrays of cell clusters expressing defined gene products will provide a suitably rapid screen to complement proteomic studies with high-quality data validating cellular function [174,175]. Advances in the field of molecular imaging [176], such as live cell imaging and better molecular probes including quantum dots [177] will enhance our ability to observe context-dependent protein interactions, functional compartmentalisation and specific interrelationships. The ability to track cells in host animals and generate increasingly elaborate tissue-like structures with tissue engineering will extend proteomic studies to intercellular interactions. Anticipated increases in computing power and bioinformatic databases will facilitate larger proteomic studies without compromising accuracy or precision. Regarding the original metaphor, proteomics is rapidly progressing from ‘‘hour-glass’’ to ‘‘clockwork’’ and now heading for ‘‘quartz accuracy’’. REFERENCES 1

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In memoriam Prof Excelentisimo Dr Jordi Sans i Sabrafen (1933–2004).

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