This action might not be possible to undo. Are you sure you want to continue?
Annu. Rev. Pathol. Mech. Dis. 2008.3:485-498. Downloaded from arjournals.annualreviews.org by Kennesaw State University on 10/07/08. For personal use only.
Raghothama Chaerkady1,2 and Akhilesh Pandey2
Institute of Bioinformatics, Bangalore 560066, India
McKusick-Nathans Institute of Genetic Medicine and Departments of Biological Chemistry, Oncology, and Pathology, Johns Hopkins University, Baltimore, Maryland 21205; email: firstname.lastname@example.org, email@example.com
Annu. Rev. Pathol. Mech. Dis. 2008. 3:485–98 First published online as a Review in Advance on October 3, 2007 The Annual Review of Pathology: Mechanisms of Disease is online at pathmechdis.annualreviews.org This article’s doi: 10.1146/annurev.pathmechdis.3.121806.151419 Copyright c 2008 by Annual Reviews. All rights reserved 1553-4006/08/0228-0485$20.00
aptamers, isotope labeling, mass spectrometer, peptide arrays, protein arrays, protein proﬁling
Detailed and comprehensive characterization of proteins is a major goal of proteomics. This goal has become more realistic today with the latest high-resolution mass spectrometers capable of faster sequencing in a high-throughput fashion and with the emergence of new techniques such as protein and peptide microarrays. A promising area for discovery is the application of these advanced mass spectrometric and other quantitative proteomic methodologies to laboratory diagnosis. This review focuses on the role of proteomics in the development of new laboratory diagnostics and the implications for routine diagnosis and monitoring of diseases.
3:485-498. 2008. Figure 1 outlines various proteomic methodologies that have the potential to be applied to laboratory diagnosis. Mech.INTRODUCTION Aptamer: RNA or double-stranded DNA molecules that bind to speciﬁc molecular targets ranging from small molecules to proteins Annu.org by Kennesaw State University on 10/07/08. Downloaded from arjournals. Proteomics can be applied to screen clinical samples for diagnosis.. Both in vivo and in vitro methods of stable isotope labeling are available for studying the dynamics of protein modiﬁcations (3). 486 Chaerkady · Pandey . Mass spectrometry (MS) has become a technique of choice for protein identiﬁcation.g. for prognosis.annualreviews. For personal use only. a number of proteomic platforms have been developed or perfected for purposes ranging from studying protein-protein interactions and posttranslational modiﬁcations to protein detection and quantitation. Liquid chromatography coupled to tandem MS is now routinely used for the sensitive identiﬁcation of complex protein mixtures (1). we discuss advances in laboratory diagnostics that could be developed on the basis of proteomics platforms.g. and for therapeutic monitoring of diseases. Rev. Over the past decade. One of the popular methods is stable isotope labeling of samples in which the relative concentration of proteins can be accurately determined by chemically identical heavy and light peptides (2). and bead-based technologies. aptamer arrays. surface-enhanced laser desorption/ionization time-of-flight) (SELDI-TOF) Direct tissue analysis for biomolecules (e.. Pathol. matrix-assisted laser desorption/ ionization time-of-flight) (MALDI-TOF) Figure 1 Different proteomic approaches used for the discovery of biomarkers for the development of a diagnostic tool. Dis. MS has rapidly emerged as the Proteomic methodologies for the development of potential lab diagnosis Mass spectrometry–based proteomics Array-based methods •Antibody arrays •Protein lysate arrays •Peptide arrays •Bead-based arrays •Aptamer arrays Imaging mass spectrometry Pattern recognition Absolute quantitation Spiking with known amount of heavy peptide Label-free methods •Spectral counting •Peak area measurements Relative quantitation •Stable isotope labeling with amino acids in cell culture (SILAC) •Isotope tagging for absolute and relative quantitation (iTRAQ) •18O labelling •Isotope-coded affinity tag (ICAT) Selective immobilization of samples on chemically modified surfaces (e. Simultaneous identiﬁcation and quantitation of proteins can be achieved by advanced MS approaches. Mass Spectrometry as a Discovery Tool for Disease Biomarkers Mass spectrometers accurately measure the mass-to-charge (m/z) ratios of ionizable compounds. Here. The application of proteomics to routine laboratory diagnosis has gained tremendous interest because of the success of proﬁling studies. whereas for applications such as protein quantitation there are other options such as protein microarrays.
annualreviews. The absolute amount of a protein in samples can be measured through the use of spiked heavy peptides as internal standards (8).1 0 0 598 1 598.31 0 602 376.25 376 378 m z –1 m z –1 372 374 m z –1 m z –1 Figure 2 Discovering biomarkers using quantitative proteomic approaches. representative mass spectra are shown. ity tags (ICATs) (5). For each method. 18 O labeling methods. the development of quantitative proteomic approaches has facilitated the application of MS to identifying biomarkers for various diseases.80 598.annualreviews.8 4 0 500 504 508 512 2 504.org by Kennesaw State University on 10/07/08.3 MS 100 MS/MS 5 510.8 507.8 502.technique of choice for the analysis of proteins and peptides.81 2 1 114. Mech. Furthermore.31 MS 100 2 1 371.3:485-498.1 100 2 600. www.75 377. One promising area is the use of label-free methods of quantitative proteomics in which the peak area for peptide ions in mass spectra are directly used as a measure of protein abundance (9).1115. Rev.1 115. including cancers.1 114. and isotope-coded afﬁnity tagging.25 372. isotope tagging for absolute and relative quantitation (6).3 1 504.8 505. In vitro labeling methods shown in this ﬁgure include isotope tagging for absolute and relative quantitation. Stable isotope labeling with amino acids in cell culture is an in vivo method for the relative quantitation of proteins. or in vitro methods such as isotope-coded afﬁnAnnu.80 599 600 601 601.25 600.3 502.75 372. Figure 2 shows how some In vivo method In vitro methods Metabolic labeling Stable isotope labeling with amino acids in cell culture (SILAC) method 1 2 3 4 5 N-terminal labeling Isotope tagging for absolute and relative quantitation (iTRAQ) method 1 2 3 4 C-terminal labeling 18 O labeling method 1 16 Labeling cysteine Isotope-coded affinity tag (ICAT) method 1 2 13 2 18 O O 12 C9 ICAT tags C9 Normal Cancer-derived cells grown in cells grown in light amino acids heavy amino acids Protein digestion 114 115 116 117 tags Protein digestion Label proteins using ICAT reagents Label peptides using iTRAQ reagents Mix Mix Protein digestion Mix Mix Protein digestion Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) Relative intensity (%) 100 LC-MS/MS LC-MS/MS LC-MS/MS 3 505. Downloaded from arjournals. Some of the commonly used quantitative proteomic methodologies include in vivo labeling using the stable isotope labeling with amino acids in cell culture method (4).3 3 116. and 18 O labeling (7).3 511.30 599.8 507.75 MS 376.org • Applications of Proteomics to Lab Diagnosis 487 . For personal use only. Dis.1 4 117. 2008. Pathol.
Several intermediary metabolic compounds in serum or urine are detected using MS to diagnose inborn errors of metabolism in children. Rev. Microorganisms produce characteristic chemical ions such as fatty acids.3:485-498. Mice bearing tumor xenografts have been used as a model proteomic platform to address heterogeneity in analysis of tissues. which are excellent Chaerkady candidates for the diagnosis of speciﬁc bacterial pathogens. robustness. For example. Another approach being tried is the use of matrix-assisted laser desorption/ionization mass spectrometry for direct tissue imaging. serum. Stable isotope labeling with amino acids in cell culture: an in vivo method for metabolic incorporation of stable isotopes into proteins by growing cells in culture media that contain heavy versions of amino acids Isotope tagging for absolute and relative quantitation: set of amine-speciﬁc isobaric tags for multiplexed relative quantitation of proteins by tandem mass spectrometry Surface-enhanced laser desorption/ionization mass spectrometry: used to selectively capture samples on a solid surface and analyze the mass spectral pattern generated by laser-induced ionization Matrix-assisted laser desorption/ ionization mass spectrometry: involves soft ionization of samples cocrystallized with a matrix.org by Kennesaw State University on 10/07/08.Annu. a precursor ion and one of its fragment ions (a prominent ion in MS/MS spectrum) are selected for quantitation. Table 1 provides a list of some of the published studies on biomarker identiﬁcation using quantitative proteomic approaches. Downloaded from arjournals. and homocysteine into their fragment ions can be monitored and quantitated by MS (15). 20). and 2-docosanol in drinking water for Mycobacterium xenopi contamination (18). For personal use only. However. Mech. Proteomic analysis of altered signaling through tyrosine kinase pathways is a strategy for identifying cancer biomarkers for diagnostic and/or therapeutic purposes. Miniaturization.. Advancement in instrumentation is essential for MS-based clinical diagnosis. a given peptide) using MS provides valuable information for localization. proﬁling. heterogeneity of samples. complexity of biomolecules. Mass spectrometric analysis of immunoprecipitated tyrosine phosphopeptides was recently used to discover aberrant activation of JAK3 kinase due to a mutation in a subset of acute megakaryoblastic leukemia cells (11). and other biological ﬂuids. Absolute quantitation approaches have been used to quantitate the actual amount of proteins in a sample.g. Mass Spectrometry as a Tool for Lab Diagnosis Mass spectrometers are already in clinical practice as a diagnostic tool in laboratories. A similar approach has been used to identify potential biomarkers for diagnosis in cerebrospinal ﬂuid from patients with Alzheimer’s disease (13). Pathol. tuberculostearic acid in sputum for pulmonary tuberculosis (17). using mass ﬁlters in tandem MS. Such proteins have been identiﬁed using the stable isotope labeling with amino acids in cell culture method from pancreatic-cancer-derived cells and conﬁrmed by Western blotting and immunohistochemical studies using tissue microarrays (10). metastasis. as shown recently for lung cancer (12. 2008. and unidentiﬁed peak patterns limit the use of such tools for broad diagnostic applications. conversion of amino acids such as phenylalanine. and immune escape have been studied in pancreatic cancers using the ICAT method (12). This is usually done by spiking known amounts of heavy synthetic peptides and quantitating signature ions in the selected-reaction-monitoring mode. Direct tissue imaging of a speciﬁc compound (e. and distribution between the tissues. Proteins associated with tumor invasion. Surface-enhanced laser desorption/ionization mass spectrometry is an attempt to diagnose diseases using mass spectral patterns as markers of diseases. In the selected-reaction-monitoring mode. and simplicity of operation are key factors in making it feasible for routine diagnostics (19. methionine. On the basis of the same 488 · Pandey . the mass-to-charge values of the ions are calculated from their time of ﬂight in the mass analyzer of the commonly used methods for quantitative proteomics can be applied to biomarker discovery. 14).annualreviews. If these technologies become robust enough. it is conceivable that direct proﬁling of tissue and serum or other biological ﬂuids such as urine or saliva could become standard practice in laboratory diagnostics. Proteins secreted by cancer cells are also attractive candidates for biomarkers. Some of the MS-based analyses include a C16:1 fatty acid in cerebrospinal ﬂuid for bacterial meningitis (16). Dis. The composition of ionizable molecules in biological samples can change in diseases and thus can be used as the basis for their diagnosis. The standardization of mass spectrometers across laboratories is also essential for the reproducibility of diagnostic tests.
CD9. SDF4. apoE. Mech. 13 C-isotope-labeled synthetic peptides of C-reactive protein were used as internal standards Standard heavy peptide from prostate-speciﬁc antigen was used to quantitate serum prostate-speciﬁc antigen in selected-reaction-monitoring mode. used to demonstrate the use of mass spectrometer as a diagnostic tool Diseases Rheumatoid arthritis Reference(s) (22) Proteomic methods Methods involving isotopic labels Prostate disease (21) Annu.3:485-498. SILAC method was used for identiﬁcation of secreted proteins as biomarkers. and insulin-like growth factor II antibodies was used to distinguish cancer from normal Autoantigen arrays Detection of serum levels of several autoantibodies Identiﬁcation of autoantibodies to citrullinated epitopes using synovial arrays as early markers of disease Protein lysate arrays Quantitation of active extracellular signal-regulated kinase in tissue lysates using phosphopeptide reference www. and ﬁbronectin receptor were some of the potential biomarkers among the proteins identiﬁed iTRAQ method was used to study dysregulated proteins in bone marrow leukemic stem cells iTRAQ method was used to identify unique biomarker proteins in cerebrospinal ﬂuid from patients with neurodegenerative disorders ICAT method was used for quantitative proﬁling of microdissected tissues for biomarker discovery 18 O-isotope Pancreatic cancer (10) Leukemia Alzheimer’s disease (55) (56) Hepatocellular carcinoma Lung cancer (57) (58) labeling method was used on low-molecular-weight proteins as biomarkers from serum of mice bearing cancer xenografts Imaging MS Direct imaging of cancer tissue using photocleavable mass-tagged secondary antibody.org by Kennesaw State University on 10/07/08. chemokines. Rev. Downloaded from arjournals.org • Applications of Proteomics to Lab Diagnosis 489 . prolactin.annualreviews. For personal use only. perlecan. and growth factors were detected A panel of leptin. osteopontin.annualreviews. cancer biomarker proteins PS-100 and HMB45 were detected using MALDI time-of-ﬂight MS Direct tissue imaging of ﬁne-needle aspirates using MALDI time-of-ﬂight Melanoma (59) Lung cancer Acute GVHD Hypertension Breast cancer Renal cell carcinoma Prostate cancer Infectious meningitis Ovarian cancer Systemic lupus erythematosus Rheumatoid arthritis Ovarian cancer (60) (61) (62) (63) (64) (65) (66) (67) (68) (69) (28) SELDI time-ofﬂight MS Serum proteomic pattern of 8 m/z species were shown to discriminate GVHD from non-GVHD Spectral patterns studied in a rat model Fine-needle aspiration and collection of intact sample from breast tissue for pattern recognition Patterns in sera of patients before and after surgery. serum amyloid alpha was identiﬁed as a potential biomarker Antibody arrays Validation of thrombospondin as a biomarker Novel cytokines.Table 1 Clinical laboratory applications of various proteomic methodologies Clinical laboratory applications Quantitation of C-reactive protein from prefractionated serum using multiple-reaction-monitoring mode. Dis. 2008. Pathol.
and aptamer arrays. isotope-coded afﬁnity tag. multiple analytes can also be simultaneously monitored by MS (multiple reaction monitoring). isotope tagging for absolute and relative quantitation.org by Kennesaw State University on 10/07/08. Antibody arrays have been used for different protein-proﬁling approaches in cancers (24). lack of precision in the immobilization of proteins onto solid surfaces could still lead to poor reproducibility and Protein Arrays for Lab Diagnosis Protein arrays are arrays of thousands of proteins immobilized on surfaces such as glass or nitrocellulose (23). which drastically increase the sensitivity and speciﬁcity of analysis using protein arrays. For personal use only. principle. Dis. However. Several types of antibody arrays have been developed to accommodate samples. among others (Figure 3). Protein arrays have been used to address various clinical diagnostic needs. In this method. graft-versus-host disease. for example. validation of molecular changes in a large number of samples and dissecting signaling pathways in different types of cancers.3:485-498. Mech. Selection and generation of speciﬁc antibodies for novel protein markers are also important processes in the development of antibody arrays.Table 1 (Continued ) Clinical laboratory applications Multiplexed analysis of serum for human papillomaviral proteins Quantitation of circulating interleukin-8 and anti-interleukin-8 autoantibodies Detection of human antidengue virus IgG in clinical serum samples Vascular endothelial growth factor was detected at concentration of physiological limit Highly speciﬁc aptamer for HIV type 1 reverse transcriptase Diseases Cervical cancer Ovarian cancer Dengue virus infection Rheumatoid arthritis. Pathol. The major limitations of antibody arrays include lower reproducibility of quantitation and the need for optimization conditions for antibodies (25). varying from whole cells and protein extracts to puriﬁed proteins. GVHD. recombinant protein arrays. lung cancer Reference(s) (70) (71) (72) (45) Proteomic methods Bead-based arrays Biosensor magnetic beads Aptamer arrays Annu. matrix-assisted laser desorption/ionization. Antibody arrays can be used for the initial screening of cross-reactivity with proteins using an entire proteome. Downloaded from arjournals. 2008. antibodies are immobilized on a solid surface and are used to capture the antigens from the solution phase. Here we discuss different types of protein arChaerkady Selected reaction monitoring: monitoring of ions with user-speciﬁed mass-to-charge values in a mass spectrometer. Antibody arrays. MALDI. Rev. iTRAQ. SELDI. The major advantage of protein arrays over MS-based methods is that multiple biomarkers as well as samples can be analyzed simultaneously. which is done to increase the selectivity and sensitivity of detection 490 · Pandey . (73) (47. The advantage of these methods is that it is possible to monitor unique isoforms of proteins because analysis is carried out at the peptide level. 74) Thioaptamers Detection of nuclear factor kappa B and AP-1 proteins SILAC. which is analogous to multiplexed quantitation in a single experiment. Antibody arrays are a relatively new platform for screening diagnostic markers for diseases. Several improved detection methods for protein arrays are now available. One of the clinical applications of this is the routine analysis of peptides derived from established biomarkers in serum (21. stable isotope labeling with amino acids in cell culture. and different cancer HIV infection West Nile virus.annualreviews. 22). surface-enhanced laser desorption/ionization. ICAT. Protein arrays with clinical applications include antibody arrays. rays and their advantages and limitations as readouts.
Proteins of interest in samples are probed using primary antibodies and detected using a number of advanced methods. Protein lysate arrays can be used for identifying altered signaling 491 www. Antibody.Antibody array Incubate various body fluids Panel of markers in (e. Downloaded from arjournals.annualreviews. Peptide array Incubate with tissue lysate Incubate biological fluid with arraycontaining peptides from a pathogen Incubate with antibodies Profiling of kinase activity in the tissue Detection of infectious diseases Recombinant protein array Incubate with tissue lysate Incubate with biological fluids (serum/cerebrospinal fluid/urine/saliva) Profiling of kinase activity in the tissue Detect autoantibodies against specific antigens Epitope mapping Figure 3 Different types of array-based approaches for lab diagnosis. Protein lysate arrays are also known as reverse-phase protein arrays in which protein lysates are spotted onto a nitrocellulose-coated glass surface instead of individual bait molecules such as antibodies. Pathol.annualreviews. and recombinant protein arrays are potentially useful tools for different laboratory diagnostics. Some of the published studies on diagnostic applications of antibody arrays are listed in Table 1. For personal use only.org • Applications of Proteomics to Lab Diagnosis . 2008.. aptamer. Mech. Protein lysate arrays. Different methods of sample collection are compatible with protein lysate arrays. peptide.g. biological ﬂuids such as serum from patient populations.org by Kennesaw State University on 10/07/08. This variability can be reduced by immobilizing a known number of antibodies/proteins by means of a ﬁxed number of oligonucleotide immobilized onto the solid surface. and internal controls can be spotted directly onto array grids. Cy3/Cy5-labeled various types of cancers proteins from serum/ cerebrospinal fluid/urine/saliva) Cytokine screening in various body fluids Incubate with body fluids followed by lectin affinity detection Membrane-proteinspecific array: incubate with cells Diagnosis and prognosis of diseases Glycoprotein biomarkers in cancer Monitioring CD antigens in cancers and infectious diseases Aptamer array Incubate with arraycontaining aptamers that recognize proteins Incubate with arraycontaining aptamers that recognize carbohydrates Aptamers for aminoglycosides Biomarkers as a signature Detection of cells based on membrane sugar Screening specific antibodies Annu. laser-captured microdissected cells. For instance. Dis. Rev.3:485-498. well-to-well and/or array-to-array variation.
wheat germ agglutinin.3:485-498. The expression of glycoproteins on the cell membrane is known to be altered in pathological conditions (30–32).g. tions).org by Kennesaw State University on 10/07/08. are resistant to nucleases and perhaps a better support for the development of aptamers as diagnostic tools (47). Mech.g. Pathol.. Aptamers (42) are singlestranded DNA or RNA oligonucleotides synthesized in a combinatorial fashion and immobilized on a solid surface through oligonucleotides. Thioaptamers. peptide synthesis is easy. unlike proteins. Many of the existing biomarkers of cancers are. Downloaded from arjournals. Peptides can be used as substrate screens to obtain the readout of kinases in cells by incubating lysates from cells/tissues. 492 Chaerkady · Pandey . Some of the biological applications of lectins include the afﬁnity puriﬁcation of glycoproteins. whereas a different lectin. Lectin arrays. Dis. the lectin concanavalin A speciﬁcally binds α-mannosyl groups. Lectin-array-based identiﬁcation of glycoconjugates is applicable to cancers and autoimmune and infectious diseases. the antigen retrieval step can also be avoided.. Rev. has a high speciﬁcity for N-acetylglucosamine-containing glycoproteins.Lectins: proteins that bind carbohydrates and carbohydratecontaining compounds. 38). can be distinguished by their carbohydrate speciﬁcity pathways. in fact. although many rounds of selection result in aptamers with higher afﬁnities. and reproducible. However. Some of the other applications include the detection of autoantibodies in sera (39) and identifying the activating signaling pathway molecules (40). Some of the detection methods are based on surface plasmon resonance imaging of aptamer-protein-antibody complexes (45) and ﬂuorescence detection of aptamerconjugated nanoparticles (46). When denatured proteins are used. blood group typing. In this method. For example. Various applications of peptide arrays include serological detection of antibodies to viral infections (e. Signaling pathways in various cancers have been studied using reverse-phase protein arrays (26–28). RNA aptamers can be immobilized on surface oligonucleotides using T4 RNA ligase (43). a new class of aptamers. Peptide arrays are made on inert solid supports. Peptide arrays have certain advantages compared with most other types of protein arrays. for example. and probing membrane glycoproteins for potential biomarker discovery may provide better diagnostic ability.annualreviews. Unlike the production of recombinant proteins (or even protein frac- Annu. EpsteinBarr virus. and hepatitis B) (37. Lectins are a group of carbohydrate-binding proteins obtained mainly from legumes. robust. For personal use only. aptamers are sensitive to nucleases and generally exhibit weak binding afﬁnity. Epitope mapping using overlapping peptide in peptide arrays can help obtain highly speciﬁc antibodies. Immobilized aptamers are used to capture speciﬁc proteins. as shown for the afﬁnity ranking of recombinant Fab fragments (41). The advantage of aptamers over antibodies is that they are physically stable. 2008. Direct analysis of cells toward this lectin afﬁnity matrix has been tested to identify the cells with the distinct proﬁle of preferred lectins (33). Peptide arrays. One of the inert surfaces used is composed of a layer of alkanethiolates on a gold surface (35). HIV. Aptamer arrays. carbohydrate moieties (e. The use of lectins as capture agents is promising because most plasma membrane and secreted proteins contain one or more carbohydrate chains. and the detection of pathogens. Detection of antibody responses to tumor antigen in patients with a speciﬁc type of cancer can be utilized for diagnostic purposes (29). protein lysates containing tumor antigen are spotted on the array and detected using sera from cancer patients. The appropriate aptamer for a given target is selected from a library of aptamers by a process called systematic evolution of ligands by exponential enrichment and phage display (44). Lectin microarrays have been used to study normal and pancreatic cancer sera for screening biomarkers (34). CA 19–9 in pancreatic cancer). The Diels-Alder reaction of alkanethiols and diene conjugates of peptides can be used for the immobilization of peptides on the chip (36).
Bead-based arrays for lab diagnosis. DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review. www. A secondary antibody linked to a reporter ion is subsequently quantitated using ﬂow cytometer. are evolving rapidly. proteomics-driven laboratory diagnostic tools will play a dominant role in clinical diagnosis. Pathol. Mech. quality control. For example. 4. We anticipate that proteomic technologies will become essential components of most clinical laboratories within the next decade and will provide diagnostic information that drives clinical decision making. Bead-based methods can also be used for the development of antibody (52) and aptamer- based (53) assays for diagnostic platforms. The development of robust protein microarrays using miniaturization methods such as microﬂuidic emulsiﬁcation (54) can potentially be applied to new diagnostic tools. it is almost certain that the use of proteomic signatures as biomarkers will gain popularity in the coming years.org by Kennesaw State University on 10/07/08. and Blood Institute. antigen-coated beads can be used to detect the autoantibodies in the serum. Dis. 2. Proteomic technologies involving novel applications of mass spectrometry. 2008. FUTURE PROSPECTS In recent years.org • Applications of Proteomics to Lab Diagnosis 493 . ACKNOWLEDGMENTS This work was supported in part by contract N01-HV-28180 from the National Heart. Rev. 3. For example. Beadbased assay systems offer an attractive alternative to antibody-based array systems. Annu.Some of the applications of aptamers include laboratory detection of cytokines (48) and infectious agents (49) as well as of therapeutic agents (50). Proteomic methodologies for the study of global protein expression and posttranslational modiﬁcations have greatly inﬂuenced clinical research. a library of aptamers or antibodies can be screened for analyzing optimal conditions for protein binding. In the future. Standardization. Although only a small subset of the human proteome is currently used in a diagnostic setting.3:485-498.annualreviews. SUMMARY POINTS 1. and sharing of data across laboratories are additional challenges for the introduction of proteomics to laboratory diagnosis. Speciﬁc color coding of beads allows a multiplexed qualitative and quantitative analysis of up to 100 different analytes. prognosis. However. Downloaded from arjournals. such as direct tissue imaging. proteomics-based discoveries have contributed tremendously to elucidating molecular alterations in human diseases. Multiplexed bead-based assays have been applied to the proﬁling of cytokines for the early detection of ovarian and head and neck cancers (51). Lung. For personal use only. and therapeutics.annualreviews. proteomics is not being applied routinely to clinical medicine at this time because proteomics tools are not robust enough for routine diagnosis.
Williamson B. Rev. Kristiansen TZ. Label-free quantitative proteomics using large peptide data sets generated by nanoﬂow liquid chromatography and mass spectrometry. 2004. Ong SE. 48:1970–80 16. Huang YN. 2003. Microbiol. Steen H. Mech. Med. 494 · Pandey . Gerber SA. Honda K. Quantitative analysis of complex protein mixtures using isotope-coded afﬁnity tags. metastasis. 2006. Dis. Pitt JJ. et al. Wang H. Biotechniques 10:65–75 12. Mol.3:485-498. 2001. Biotechnol. Proteomics 5:1338–47 10. Clin. discussion 73–80 14. Pandey A. Cheung SW. Gelb MH. Biomarker discovery from pancreatic cancer secretome using a differential proteomic approach. Ross PL. Infect. et al. Parker K. Kuick R. 2002. Turecek F. Pan S. Activating alleles of JAK3 in acute megakaryoblastic leukemia. Pandey A. Mann M. Chen R. 2007. as a simple and accurate approach to expression proteomics. Cell Proteomics 1:376–86 5. Proteomics 5:157–71 11. Quinn JF. Eng J. and immunologic escape. Cheng AF. Absolute systems biology-measuring dynamics of protein modiﬁcations.org by Kennesaw State University on 10/07/08. Aebersold R. Science 312:212–17 3. 2002. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Eggington M. Discovery of cancer biomarkers through the use of mouse models. Goodlett DR. Kuwabara H. Pancreatic cancer proteome: the proteins that underlie invasion. Chan CY. et al. Schnolzer M. 2005. Mol. et al. Walters DK. Alzheimers Dis. Poon D. J. Describes the biomarker discovery in serum to detect proteins secreted speciﬁcally by tumor cells from xenografts. 1987. Ono M. Hendrickson RC. The absolute quantiﬁcation strategy: a general procedure for the quantiﬁcation of proteins and post-translational modiﬁcations. Pathol. Nat. Oo KT. Mol. Comprehensive screening of urine samples for inborn errors of metabolism by electrospray tandem mass spectrometry. Cell.LITERATURE CITED 1. et al. Shitashige M. 2005. Aebersold R. Diagnosis of pulmonary tuberculosis by detection of tuberculostearic acid in sputum by using gas chromatography-mass spectrometry with selected ion monitoring. 70:437–73 2. Methods 35:265–73 9. J. Blagoev B. Tyner JW. Rapid diagnosis of bacterial meningitis by the detection of a fatty acid marker in CSF with gas chromatographymass spectrometry and selected ion monitoring. Mol. French GL. Gerber SA. Zhang J. Downloaded from arjournals. For personal use only. 10. Gu TL. Jedrzejewski P. et al. 156:356–62 Chaerkady Annu. 7:125–33. et al. 31:21–26 17. The stable isotope labeling with amino acids in cell culture method was used to discover secreted proteins overexpressed by a pancreatic cancer cell line. Reddy R. Stable isotope labeling by amino acids in cell culture. Kristensen DB. Quantitative proteomics of cerebrospinal ﬂuid from patients with Alzheimer disease. Gygi SP. Cancer Lett. 2008. Isobe T. Donohoe S. Kirkpatrick DS. 17:994–99 6. 21:467–70 4. 1999. 2006. Rist B. Webb CP. 2006. 2006. Monsma DJ. Protease-catalyzed incorporation of 18O into peptide fragments and its application for protein sequencing by electrospray and matrix-assisted laser desorption/ionization mass spectrometry. Mercher T. Yi EC. Cell. Electrophoresis 17:945– 53 8. Iwahori A. 1990. Kratchmarova I. Domon B. Kaye JA. Mass spectrometry and protein analysis. Analysis of proteins and proteomes by mass spectrometry. Cheung SW. Annu. Gygi SP. Cell. Lehmann WD. et al. 1996. Chan CY. Trends Biotechnol. Rev. 14. J. Peskind E. Chem. Misek DE. Dis. French GL. Chang R. Raghothama C. 2005. Biochem.annualreviews. SILAC. 249:40–48 15. Kahler SG. Gronborg M. Proteomics 3:1154–69 7. Marchese JN. Gastroenterology 129:1187–97 13. O’Hare T.
Sheehan KM. 28. Expression of α1-6 fucosyltransferase in rat tissues and human cancer cell lines. Biotechnol. Liao H. Miyoshi E. 2005. Badman ER. 1992. 1997. 25:55–78 36. 50:1907–20 26. et al. 2004. Proteome Res. Environ. Dis. et al.annualreviews. 1999. Jaresova M. Cell. J. Downloaded from arjournals. Whitesides GM. Demonstration of reverse-phase protein microarrays identifying molecular alterations in metastatic ovarian cancer using phosphorylationspeciﬁc antibodies. Proteomics 4:1175–86 23. Cell. Clinical aspects of glycoprotein biosynthesis. Kron SJ. Oncogene 20:1981–89 28. Brenner DE. J. 2006. 1993. Cooks GR. Cooks RG. Rapid Commun. Klee GG. 2004. Hori M. Int. Describes the experimental strategies used in a variety of antibody arrays and their applications in cancer research. Wu J.annualreviews. 495 . Grubb RL. Cancer 72:1117– 21 31. Clin. Chen T. 2003. Mol. Alugupalli S. Evaluating sandwich immunoassays in microarray format in terms of the ambient analyte regime. Biophys. 58:3538–41 19. Using self-assembled monolayers to understand the interactions of man-made surfaces with proteins and cells. Huh JH. For personal use only. 2005. Karl J. 2001. Development of natural protein microarrays for diagnosing cancer based on an antibody response to tumor antigens. Granovsky M. Biomol. 2004. Screening of glycosylation patterns in serum using natural glycoprotein microarrays and multi-lectin ﬂuorescence detection. Eppinger J. Antibody arrays in cancer research. J. Annu. Printing proteins as microarrays for high-throughput function determination. J. Zolg W. Lu Y. Calvert VS. Rev. Characterization of a serial array of miniature cylindrical ion trap mass analyzers. Clin. Mol. Patwa TH. Zheng T. Lubman DM. Use of reverse phase protein microarrays and reference standard development for molecular network analysis of metastatic ovarian carcinoma. Kuhn E. 20:270–74 www. 13:2444–49 21. Goodmanson MK. Mass Spectrom. Haab BB. Calvert VS. Mrksich M. Bichsel VE. Proteomics 3:2142– 46 27. Anal. Warren CE. Lab. Houseman BT. Rev. Ouyang Z. Reverse phase protein microarrays which capture disease progression show activation of prosurvival pathways at the cancer invasion front. Fishman D. Soc. 35:659–71 20. J. Sci. Signal pathway proﬁling of prostate cancer using reverse phase protein arrays. Proteomics 4:346–55 29. Peelen D. Anderson MA. Brinker A. Quantiﬁcation of C-reactive protein in the serum of patients with rheumatoid arthritis using multiple reaction monitoring mass spectrometry and 13C-labeled peptide standards. Schreiber SL. Mech.org • Applications of Proteomics to Lab Diagnosis Annu. Peptide chips for the quantitative evaluation of protein kinase activity. 1999. Mass Spectrom. Madoz-Gurpide J. MacBeath G. Miniature mass analyzers. Absolute quantiﬁcation of the model biomarker prostate-speciﬁc antigen in serum by LC-MS/MS using protein cleavage and isotope dilution mass spectrometry. Okon R. Noda K. Am. Kay EW. Larsson L. Microbiol. Simone NL. Proteomics 4:377–83 25. Wulkuhle JD. 2000. 2008. 24. Smith LM. Protein glycosylation in development and disease. Kuick R. 2004.org by Kennesaw State University on 10/07/08. Appl. Guild B. Science 289:1760–63 24. Saviranta P. Warashina M. Charboneau L. et al.18. Chem. Chem. Crit. Paweletz CP. Mrksich M. Chem. Misek DE. 2005. Lectin arrays for proﬁling cell surface carbohydrate expression.3:485-498. Nat. Geierstanger BH. Linehan WM. Simeone DM. Application of gas chromatography-mass spectrometry for rapid detection of Mycobacterium xenopi in drinking water. 3:644–52 22. 3:261–67 30. Brockhausen I. Muddiman DC. Struct. Dennis JW. Badman ER. 127:9982–83 34. Zhao J. Paweletz CP. Barnidge DR. Slosarek M. Taniguchi N. et al. Qiu J. Hayashi N. Pathol. Rev. 1996. 30:65–151 33. Proteome Res. 2002. Bioessays 21:412–21 32. 2000. Uozumi N. 78:6411–21 35.
Proteomics 5:2402– 11 42. Acad. Roberts JD. Li X. J. 279:49206–13 41. Nucleic acid approaches for detection and identiﬁcation of biological warfare and infectious disease agents. Brocks B. Corn RM.3:485-498. Gannot G. 1082:116–19 48. Mech. Nucleic Acids Res. Malingue F. Reece LM. 2004. 22:1423–28 51. Beasley DW. Hamula CL. In vitro selection of RNA molecules that bind speciﬁc ligands. Ann. Lab Chip 4:310–15 496 Chaerkady Annu. Yang X. Lapedes AS. Biol. Stoll D. 2006. Rev. Dis. Deﬁning critical residues in the epitope for a HIV-neutralizing monoclonal antibody using phage display and peptide array technologies. Cancer Epidemiol. Gillespie JW. Linkov F. 2005. Nilsson P. Rennert P. 2004. Biotechniques 35:862–69 50. Downloaded from arjournals. Immunoﬂuorescence assay and ﬂow-cytometry selection of bead-bound aptamers. Early detection of head and neck cancer: development of a novel screening tool using multiplexed immunobead-based biomarker proﬁling. 2003. Medley CD. Selection of thioaptamers for diagnostics and therapeutics. 1993. An integrated digital microﬂuidic lab-on-a-chip for clinical diagnostics on human physiological ﬂuids. Benos PV. 2007. 323:701–27 45. Methods 38:324–30 49. Chem. 2007. Le XC. Diks SH. Zhao X. Yang X. J. Y. Tang Z. Pamula VK. Chem. Guthrie JW. Biomarkers Prev. Corn RM. Marrangoni A. Uhlen M. Tan W. Schwenk JM. Olivier C. Anal. Szostak JW. 2003.org by Kennesaw State University on 10/07/08. Velikokhatnaya L. Chem. 2008. 34:6416–24 44. Poetz O. Fisher R. Gattuso A. Mol. Peptide-protein microarrays for the simultaneous detection of pathogen infections. Mol. Schwenk JM. 2002. Jellis CL. Lofton C. Ostendorp R. 79:3075–82 47. et al.37. Assays for cytokines using aptamers. Desmet R. et al. Nucleic Acids Res. Bioconjug. Hommes DW. 2007. 2006. Bouzidi A. Gene 137:63–68 39. Lindberg J. Determination of binding speciﬁcities in highly multiplexed bead-based assays for antibody proteomics. 16:102–7 52. Nature 346:818–22 43. For personal use only. Volk DE. O’Neil DJ. Smith JE. Kinome proﬁling for studying lipopolysaccharide signal transduction in human peripheral blood mononuclear cells. Ivnitski D. Bassett SE. Lee HJ. 2005. Pitoc GA. Shangguan D. Tangrea MA. 79:1082–88 46. Li Y. Fair RB. Li Y. Antidotemediated control of an anticoagulant aptamer in vivo. Stormo GD. Anal. Mol. Detection of protein biomarkers using RNA aptamer microarrays and enzymatically ampliﬁed surface plasmon resonance imaging. Nat. Yurkovetsky Z.annualreviews. et al. 2006. Boyd J. et al. O’Toole T. Lisovich A. Diagn. 15:307– 16 38. Zhang H. Salinas P. Biol. Nimjee SM. Layered peptide arrays: high-throughput antibody screening of clinical samples. Cradick TJ. Srinivasan V. et al. Calidonna M. Kok K. et al. et al. · Pandey . Prow TW. Probabilistic code for DNA recognition by proteins of the EGR family. Wallis BS. van Dijken P. 7:427–36 40. Cell. Aptamerconjugated nanoparticles for the collection and detection of multiple cancer cells. N. Lee HJ. 31:e54 54. Biotechnol. White RR. Rusconi CP. Chem. 2007. J. Erickson HS. Ellington AD. Fabrication and characterization of RNA aptamer microarrays for the study of protein-aptamer interactions with SPR imaging. Duburcq X. 1990. 2004. Schlicht R. Sci. Wang H. Sundberg M. 2004. et al. Protein microarrays for antibody proﬁling: speciﬁcity and afﬁnity determination on a chip. Pathol. et al. Proteomics 6:125–32 53.
J. Ai JH. 12:5142–50 61. et al. Koedel U. et al. Haab BB. Hood BL. Patterns of protein expression in infectious meningitis: a cerebrospinal ﬂuid protein array analysis. Invest. 2007. Lokshin AE. Fine-needle aspiration in PreservCyt: a novel and reproducible method for possible ancillary proteomic pattern expression of breast neoplasms by SELDI-TOF. Fusaro V. Rev. Haanen JB.org by Kennesaw State University on 10/07/08. Clin. Tan YX. Schellens JH. 16:1221–30 59. Gonzalez A. Exp. Winans M. Lai Y. Lucas DA. J. 2006. Rapid Commun. Delbosc S. 2006. Hematol. Engwegen JY. 2005. Sci. 9:293–348 57. Accurate diagnosis of acute graft-versus-host disease using serum proteomic pattern analysis. Proteomics 6:5169–82 56. Selective proﬁling of proteins in lung cancer cells from ﬁne-needle aspirates by matrixassisted laser desorption ionization time-of-ﬂight mass spectrometry. Schwartz P. Seshi B. Audard V. 60. 357:331–41 63. Mass Spectrom. USA 102:7677–82 68. Hueber W. Mehra N. Neuroimmunol. Zhou J. Bruce B. Meilhac O. Validation of SELDI-TOF MS serum protein proﬁles for renal cell carcinoma in new populations. 2007. Mass tags and antibodies are used for speciﬁc cancer marker detection. Mol. Dis. et al. Mangold L. Leverenz JB. Circulating IL-8 and anti-IL-8 autoantibody in patients with ovarian cancer. 497 . Kim G. Mod. Southern EM. Pﬁster HW. Rheum. 2006. Quinn JF. Protein array autoantibody proﬁles for insights into systemic lupus erythematosus and incomplete lupus syndromes. et al. Tomooka BH. 34:796–801 62. Kidd BA. 2006. Mech. Clin. et al. Acad. et al. J. 2007. Proc. Cancer cell proﬁling using matrix-assisted laser desorption/ ionization time-of-ﬂight analysis of cancer cells from ﬁne-needle aspirates. Sporer B. Partin AW. Natl. 147:60–70 69. 87:161–72 65. Methods Mol. Quantitative analysis of the low molecular weight serum proteome using 18O stable isotope labeling in a lung tumor xenograft mouse model.55. 2007.org • Applications of Proteomics to Lab Diagnosis Annu. 21:823–29 60. 2006. Li QZ. Am. et al. An integrated approach to mapping the proteome of the human bone marrow stromal cell. Marrangoni AM. Chan KC. Biol. Srinivasan R. Visintin I. Lundqvist A. Kastenbauer S. Cancer Res. Lab. 2005. Immunol. 2005. Branch V.annualreviews. 2004. Clin. 164:134–39 67. 52:2645–55 70. Franceschi S. Downloaded from arjournals. Soc. 102:244–51 www. Izbicka E. Michel JB. Arthritis. 2005. Lee BJ. et al. Nieland JD. Massion PP. et al. Shafer MW. Thiery G. Velikokhatnaya L. followed by matrix-assisted laser desorption/ ionization MS imaging of tissues from normal pancreas and metastatic melanoma. Zhao H. 51:1845–53 71. Alzheimers Dis. Killian JK. Multiplex human papillomavirus serology based on in situ-puriﬁed glutathione s-transferase fusion proteins. Zhou H. Accurate qualitative and quantitative proteomic analysis of clinical hepatocellular carcinoma using laser capture microdissection coupled with isotope-coded afﬁnity tag and two-dimensional liquid chromatography mass spectrometry. Gynecol. Blonder J. Exp. Cardiovascular biomarker discovery by SELDITOF mass spectrometry. Waterboer T. Angele B. Mass Spectrom. Prostate 67:255–67 66. Carr-Johnson F. Michael KM. Bonfrer JM. Chaurand P. Li C. et al. Wandstrat AE. Mobley JA. For personal use only. Jankovic J. Pathol. Multiplex target protein imaging in tissue sections by mass spectrometry: TAMSIM. Landsittel D. Lovell MO. Pathol. Shchepinov MS. et al. et al. 2008. Serum protein markers for early detection of ovarian cancer. Cell. 2005. Daniels J. Detection of biomarkers with a multiplex quantitative proteomic platform in cerebrospinal ﬂuid of patients with neurodegenerative disorders. Chem. Antigen microarray proﬁling of autoantibodies in rheumatoid arthritis. Audebourg A. Hong Y. Antibody array proﬁling reveals serum TSP-1 as a marker to distinguish benign from malignant prostatic disease.3:485-498. Mor G. Proteomics 3:399–409 58. Oncol. McIntosh M. Sehr P. Abdi F.annualreviews. 2007. 2004. Amann JM. 59. Fowler LJ. 17:1012–20 64.
Foley J. Pavski V. Rev. Su Z.annualreviews. Immunol. et al. For personal use only. Harris E. J. Rabbani ZN. 2001. Chem. Methods 314:21–29 73. 34:3577–84 Annu. Le XC. Liu Y.3:485-498. Downloaded from arjournals. Nucleic Acids Res. Aytur T. Anal. Beatty PR. Detection of human immunodeﬁciency virus type 1 reverse transcriptase using aptamers as probes in afﬁnity capillary electrophoresis. Mi J. Dis.org by Kennesaw State University on 10/07/08. Mech. 498 Chaerkady · Pandey . H1 RNA polymerase III promoterdriven expression of an RNA aptamer leads to high-level inhibition of intracellular protein activity. A novel magnetic bead bioassay platform using a microchip-based sensor for infectious disease diagnosis. 2008. Anwar M. 2006.72. Zhang X. Pathol. 73:6070–76 74. 2006. Boser B.
annualreviews.3:485-498. For personal use only. Evan Sadler p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 249 Anti-Inﬂammatory and Proresolving Lipid Mediators Charles N. Cornell. Weaver. R. Nielsen p p p p p p p p p p p p p p p p p p p p p p 67 The Inﬂammatory Response to Cell Death Kenneth L. Rock and Hajime Kono p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 99 Molecular Biology and Pathogenesis of Viral Myocarditis Mitra Esfandiarei and Bruce M. McManus p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 127 Pancreatic Cancer Anirban Maitra and Ralph H. Downloaded from arjournals. 2008.Annual Review of Pathology: Mechanisms of Disease Contents Annu. Tao Lu. Cheang. Stephanie Yacoubian.U. and Torsten O. Neal Smith. Serhan. Volume 3. Dis. Mech.org by Kennesaw State University on 10/07/08. and Rong Yang p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 279 Modeling Morphogenesis and Oncogenesis in Three-Dimensional Breast Epithelial Cultures Christy Hebner. Marchesi p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1 Molecular Mechanisms of Prion Pathogenesis Adriano Aguzzi. Matt van de Rijn. Colvin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 189 Metastatic Cancer Cell Marina Bacac and Ivan Stamenkovic p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 221 Pathogenesis of Thrombotic Microangiopathies X. Valerie M. and Patrick Loerch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 41 Gene Expression Proﬁling of Breast Cancer Maggie C. and Robert B. Rev. and Mathias Heikenwalder p p p p p p p p p p p p p p p p p p p p 11 The Aging Brain Bruce A. Hruban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 157 Kidney Transplantation: Mechanisms of Rejection and Acceptance Lynn D. Pathol. Long Zheng and J. Yankner. and Jayanta Debnath p p p p p p p p p p p p p p p p p p p p p p p p p p p p 313 v . Christina Sigurdson. 2008 The Relevance of Research on Red Cell Membranes to the Understanding of Complex Human Disease: A Personal Perspective Vincent T.
and T. The Osteoclast: Friend or Foe? Deborah V. Morens p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 499 Airway Smooth Muscle in Asthma Marc B. Walshe and Patricia A. Dis. Pear.The Origins of Medulloblastoma Subtypes Richard J. Peter Walter. Hershenson. Mech. Melanie Brown. For personal use only. Heinrich p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 557 Notch Signaling in Leukemia Jon C. Corless and Michael C.annualreviews. Aster. Blanca Camoretti-Mercado. and Stephen C. Warren S.org vi Contents . 2008.S.3:485-498. Rev. Benedict Yen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 399 Autophagy: Basic Principles and Relevance to Disease Mondira Kundu and Craig B. Ellison p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 341 Molecular Biology and Pathology of Lymphangiogenesis Terhi Karpanen and Kari Alitalo p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 367 Endoplasmic Reticulum Stress in Disease Pathogenesis Jonathan H.org by Kennesaw State University on 10/07/08. Downloaded from arjournals. Taubenberger and David M. D’Amore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 615 Indexes Cumulative Index of Contributing Authors. Lin. Gilbertson and David W.annualreviews. Pathol. Blacklow p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 587 The Role of Hypoxia in Vascular Injury and Repair Tony E. Teitelbaum p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 457 Applications of Proteomics to Lab Diagnosis Raghothama Chaerkady and Akhilesh Pandey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 485 The Pathology of Inﬂuenza Virus Infections Jeffrey K. Thompson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 427 Annu. and Julian Solway p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 523 Molecular Pathobiology of Gastrointestinal Stromal Sarcomas Christopher L. Novack and Steven L. Volumes 1–3 p p p p p p p p p p p p p p p p p p p p p p p p p p p 645 Cumulative Index of Chapter Titles. Volumes 1–3 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 647 Errata An online log of corrections to Annual Review of Pathology: Mechanisms of Disease articles may be found at http://pathol.