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10.3109/13506129.2011.630762

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Amyloid, 2011; 18(4): 177182Copyright 2011 Informa UK, Ltd.ISSN 1350-6129 print/ISSN 1744-2818 onlineDOI: 10.3109/13506129.2011.630762

Amyloidoses are characterized by the presence of extracellular amyloid deposits, constituted by fibrillar aggregates of misfolded proteins. Despite the similar morphologic appearance of fibrils, at least 28 different proteins have been detected as causative agents of human amyloidoses, 14 of which associated with systemic forms. Unequivocal typing of the amyloid deposits is a key step in the management of these diseases. Existing drawbacks of traditional, immunohistochemistry-based techniques have driven the search for alternative solutions for direct amyloid typing. Proteomics indicates the comprehensive study of the proteins in a biological sample, centered on analysis by mass spectrometry. The great potential of this approach in describing the composition of amyloid deposits and in studying the molecular features of the amyloidogenic precursors has become immediately clear and the introduction of proteomics in the clinical practice has revolutionized the field of amyloid typing. This review provides a critical overview of the various approaches that have been proposed in this specific context, along with a brief description of the proteomic methods for assessment of the circulating amyloidogenic proteins.

Keywords: Proteomics, amyloid typing, mass spectrometry

Abbreviations: MS, mass spectrometry; FFPE, formalin-fixed paraffin-embedded; SAP, serum amyloid P; APOE, apolipoprotein E; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; MudPIT, multidi-mensional protein identification technology; 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis; pI, isoelectric point; MALDI, matrix-assisted laser desorption/ionization; IMS, imaging mass spectrometry.

Introduction

The common pathogenic trait behind the class of diseases termed amyloidoses is the presence of extracellular amyloid deposits, constituted by fibrillar aggregates of misfolded

proteins [1,2]. Amyloid deposits can be systemic or localized in specific sites. Despite the similar morphologic appearance of fibrils, at least 28 different proteins have been detected as causative agents of human amyloidoses, 14 of which are asso-ciated with systemic forms [3]. In the latter, fibrils originate from circulating proteins that are transported to the target organs through the bloodstream. Besides the principal fibril-lar protein, minor amounts of other species are invariably associated to the amyloid fibrils, resulting in a complex and heterogeneous molecular composition of the deposits [47]. All amyloid fibrils share common ultrastructural and tincto-rial properties; in particular, the display of green birefringence under polarized light upon Congo red staining is a specific diagnostic marker.

Amyloid formation leads to cell toxicity and organ dysfunc-tion, translating in severe and complex clinical pictures. In systemic amyloidoses, the clinical course, treatment and prog-nosis are critically dependent on the type of amyloidogenic protein. Thus, amyloid typing is a key step in the management of these diseases. The relative prevalence of the various types of amyloidoses varies across different geographical regions. The most common systemic form in Western countries is light chain (AL) amyloidosis, a sporadic disease caused by deposi-tion of misfolded monoclonal immunoglobulin light chains. Hereditary forms (consequent to mutations in genes coding for amyloidogenic proteins) or reactive amyloidosis (associ-ated to chronic inflammatory conditions), however, can be observed in selected areas or patient populations. Treatment differs substantially between the various forms, ranging from chemotherapy in AL to liver transplantation in transthyretin (ATTR) amyloidosis. It is thus clear how misdiagnosis can lead to catastrophic therapeutic errors. Despite this hetero-geneity, the various forms have overlapping manifestations, which make their differentiation impossible on a clinical basis, without the auxilium of laboratory and pathology tech-niques. The diagnostic workflow requires the combined use of multiple approaches for demonstrating the presence of the

REVIEW ARTICLE

Proteomic typing of amyloid deposits in systemic amyloidoses

Francesca Lavatelli1 & Julie A. Vrana2

1Amyloidosis Research and Treatment Center, Fondazione IRCCS Policlinico San Matteo and University of Pavia, Italy and 2Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA

Correspondence: Francesca Lavatelli, MD, Amyloid Treatment and Research Center, Fondazione IRCCS Policlinico San Matteo, P.le Golgi 19, 27100 Pavia, Italy. Tel: +39 0382 502994. Fax: +39 0382 502990. E-mail: francesca.lavatelli@unipv.it

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amyloidogenic precursor, detecting DNA mutations associ-ated with familial forms and identifying the nature of the amy-loid fibrils. However, misdiagnosis is a well known potential pitfall [8,9]. Direct analysis of fibrils from affected tissues is the conclusive strategy for unequivocal amyloid typing. In the clinical setting, this has been traditionally done using immu-nohistochemical methods. However, immunohistochemistry has a number of drawbacks in the context of amyloid typing, which can make it unreliable.

Proteomics is the term used for indicating the comprehen-sive study of the protein constituents of a biological sample, centered on protein analysis and identification by mass spec-trometry (MS). A peculiar feature of proteomics is that pro-teins can be analyzed without the need of specific antibodies or of prior knowledge of the sample composition. Proteomics have been applied to the field of amyloidoses rather recently, but the great potential of this approach in describing the com-position of amyloid deposits and in studying the molecular features of the amyloidogenic precursors has become imme-diately clear.

The introduction of proteomics in the clinical practice has revolutionized the field of amyloid typing. This review pro-vides a critical overview of the various approaches that have been proposed for amyloid typing, along with a brief descrip-tion of the proteomic methods for assessment of the circulat-ing amyloidogenic proteins.

Proteomics for amyloid typing

The development of MS-based methods for amyloid typ-ing has been driven by the need for unbiased identifica-tion of the protein constituents of the deposits [813]. Immunohistochemistry and immunoelectron microscopy have been extensively used for this purpose. However, rec-ognized drawbacks of antibody-based methods in amyloid typing exist [1218]. Potential reasons for failure are: a) immunological methods require a-priori hypotheses on the nature of amyloid, and the typing depends on the availability and quality of the antibodies; b) contamination from plasma proteins can lead to non-specific background staining and misinterpretation of results; c) fibrillar proteins are known to be extensively structurally modified compared to the soluble precursor; in particular, truncated forms are typically found. The fragments may not contain the epitopes recognized by the antibody; d) the conformation of the deposited proteins is altered; this may impair epitope binding.

To obtain a definitive diagnosis, the amyloid contained in biopsy-derived specimens must be subjected to direct chemical analysis. Several proteomic approaches for amyloid typing have been developed, either on formalin-fixed paraffin- embedded (FFPE) or on fresh, unfixed specimens. Most methods have been optimized for analyzing minute amounts of material, such as those obtained by fine needle aspiration or tissue sections mounted on pathology slides. A number of fea-tures differentiate the various approaches, including: (1) the tissues on which the analysis can be performed; (2) the spe-cific tissue handling requirements (e.g. fixation vs freezing); (3) the methods for sample processing and protein extraction

(e.g. analysis of whole tissue vs selected amyloid areas); (4) the methods of protein separation (either gel free or gel based); (5) the methods and instruments for MS data acquisition, algorithms for protein identification, and diagnostic interpre-tation of results.

All the described approaches were shown to provide reli-able amyloid classification on the published patients series: a brief overview of the applicability and unique features of the various procedures is provided below.

Proteomic identification of deposits in tissues sectionsSeveral cutting edge techniques have been developed over the last five years that have been shown to be useful in identifying amyloid fibril composition in tissue sections. Recent devel-opments using either macrodissection or laser microdissec-tion of Congo Red stained FFPE tissue followed by trypsin digestion, tandem MS and bioinformatics can now ascertain the major amyloid types such as ATTR, AL, AA [19,20] and also provide accurate identification of more rare amyloid types such as AGel [21], AApoAI [19,22], AApoAIV [23], AH [24], ALect2 [25,26], AIns [27], ALys [19,28] and their variants [21,22]. This methodology is becoming a new clinical standard for amyloid typing because it provides much more information than that provided by antibody-based techniques. By using bioinformatic software, instead of antibodies, most of the abundant proteins within the dissected amyloid deposit can be detected. Since amyloid may contain multiple fibril types the proteome profile also allows the predominant fibril type, which is causing the disease, to be accurately determined. The absence of other amyloid fibril proteins is also taken into consideration during analysis. Figure 1 illustrates an example of amyloid types diagnosed in clinical specimens using pro-teomic profiles (ATTR, AA, ALect2, AL- and AL-). In addi-tion to proteins involved in amyloid such as serum amyloid P (SAP) and apolipoprotein E (APOE), the profile also identi-fies tissue specific proteins. As our understanding and char-acterization of amyloid matrix proteins [21,24,30] and their post translational modifications [31,32] increase, the ability to individually target these proteins therapeutically will also increase. Although the tissue preparation and LC-MS/MS protocols varied slightly between laboratories, in all cases ref-erenced above the proteomic analyses successfully identified both common and rare amyloid types. The current clinical test for amyloid typing (performed since 2008) at the Mayo Clinic has provided over 2500 patient diagnoses and over the course of several years has identified 18 different amyloid types using proteomic methodology.

Proteomic identification of deposits in tissue biopsiesProteomics has also proved successful in amyloid typing on fresh fat aspirates, without dissection of the amyloid areas [18,19,3335]. The characterization of the whole proteome of subcutaneous fat, based on MudPIT analysis (Multidimensional Protein Identification Technology) has recently been described [35]. To optimally resolve the vari-ous tissue proteins, a powerful separation procedure, based on two-dimensional chromatography, has been applied. This technique allowed identifying hundreds of proteins in each

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tissue sample, both intra- and extracellular. Identification of the causative amyloid proteins requires the comparison with a control tissue reference map and is based on the assumption that deposited proteins should be overrepresented in patients.

A key preparative step is sample washing prior to protein extraction, to remove blood contaminants. The average pro-teomic profile of non-affected tissue has been generated, to be used for a semiquantitative differential analysis, using dedicated software [36]. Besides the amyloid proteins, the dif-ferential analysis has allowed identification of other proteins up-regulated in patients, most of which are already known to associate with amyloid deposits (Table I). Since traces of multiple amyloid proteins are commonly detectable in all samples, due to residual blood contaminants, an algorithm for MS-based amyloid classification has been introduced. The classification algorithm and the comparison with the control group are important innovations in the perspective of increas-ing the reliability of typing and eliminating the confounding effects of blood contaminants.

Proteomic typing of amyloidoses based on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)This approach has been developed for diagnostic typing of amyloid deposits in unfixed abdominal subcutaneous fat aspi-rates [18,3739]. The method is based on protein separation by 2D-PAGE, according to isoelectric point (pI) and molecu-lar weight, prior to MS analysis. Samples require to be frozen immediately after acquisition, to preserve protein integrity; this analysis cannot be applied to FFPE specimens. This strat-egy is based on the assumption that the presence of abnormal proteinaceous deposits should translate in novel protein spots visible on the 2D gels, in comparison with the corresponding control maps, which can thus be isolated, analyzed by MS and identified, leading to amyloid characterization. The different types of amyloid deposits originate distinct and characteris-tic 2D-PAGE maps. The presence of amino acid variants in proteins responsible for hereditary forms can be assessed by MS; additionally, if the amino acid substitution changes the pI of the protein, the variant and wild type proteins have

Figure 1. Representative scaffold readout of proteomic profiles for five cases of amyloidosis by spectral number (top 30 proteins are shown) Results were as follows, Patient 1: ATTR, from synovium biopsy; Patient 2: AA, from kidney biopsy; Patient 3: ALect2, from liver biopsy; Patient 4: AL-, from stomach biopsy; Patient 5: AL-, from cardiac biopsy. MS raw data files were queried using three different algorithms (Seaquest, Mascot and X!Tandem) and the results were combined and assigned peptide and protein probability scores in Scaffold (Proteome Software, Portland, OR). For each case a list of proteins based on pep-tide identification (peptide identifications were accepted if established at >90% probability as specified by the Peptide Prophet algorithm) were accepted.

Table I. Up-represented proteins in adipose tissue samples of patients with systemic amyloidosis (AL, AL, ATTR and AA), analyzed via MudPIT-based proteomics.

Up-represented proteinsNo. Accession Referencea AL patients AL patients ATTR patients AA patients

1 134167 SAA ++2 18655500 LC ++ 3 230651 TTR ++ 4 106659 LG ++ 5 93163358 Apo-AIV + + 6 4557325 Apo-E + + 7 42740907 Clusterin + + 8 24212664 HSPG + 9 88853069 Vitronectin + +

10 5454086 SRPX + 11 576259 SAP + + Patients have been grouped according to amyloid type. ++ up-represented proteins in all patients, + up-represented proteins in more than 50%, but less than 100% of pa-tients; non up-represented proteins. In AL and AL patients, peptides of both constant and variable regions of immunoglobulin light chains were identified; LC and LC were the proteins to which peptides of constant region were attributed (Adapted from Brambilla et al, Blood 2011).aNCBInr Reference.Apo-AIV, apolipoprotein A-IV; Apo-E, apolipoprotein E precursor; Clusterin, clusterin isoform 2 preproprotein; HSPG, basement membrane-specific heparan sulfate proteo-glycan core protein; LC , chain L, crystal structure of tissue factor in complex with humanized Fab D3h44; LC , Ig chain human; SAA, serum amyloid A protein precursor; SAP, chain A, the structure of pentameric human serum amyloid P component; SRPX, Sushi-repeat-containing protein, sushi repeat-containing protein SRPX isoform 1; TTR (Transthyretin), chain A, structure of prealbumin, secondary, tertiary and quaternary interactions determined by Fourier refinement at 1.8 Angstroms; Vitronectin, vitronectin precursor.

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distinct migration patterns on the gel. Compared to the other approaches, the gel-based one provides direct visualization of the tissue proteome, allowing an estimation of the relative amounts of the various species. It is also unique in its ability to finely dissect and separate all the charge isoforms and frag-ments of the deposited amyloidogenic proteins. As mentioned, as all approaches based on the analysis of unfractionated tis-sue, this method is dependent on the availability of control reference maps for each specific tissue under examination.

Amyloid typing by imaging MSThis technique targets the identification of proteins directly from the tissue mounted on a slide, allowing the tissue to remain intact and maintaining accurate protein and peptide tissue distribution [40]. In this method, matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is performed on a tissue section to establish the pro-tein and peptide spatial distribution. First matrix is spotted on the tissue and subsequently ionized in a discrete geo-metrical pattern [41]. Subsequently the mass spectra obtained from each spot on the tissue section contains the molecular weight and intensity information of the proteins present at that position. This information can be plotted to produce m/z specific images or ion density maps [42]. Then a serial tissue section is spotted with trypsin using an automated chemi-cal ink-jet printer to carry out an in situ digestion which is followed by peptide sequencing of a predicted fragment by MALDI MS/MS. The proteins are identified using the Mascot (Matrix Science, Boston MA) searching algorithm. In the ref-erenced example, IMS analysis resulted in the identification of arginine-containing peptides that matched predicted tryptic peptides of serum amyloid A [40]. This technique, of direct identification of proteins from tissue using MALDI MS, can provide a proteomic map over a whole tissue section.

Specific issues in applying proteomics to amyloid typing

Specific caveats exist in using proteomics as diagnostic instru-ment in amyloidoses. A first issue is related to standardiza-tion of the techniques. Instead of being dependent on good histology or immunohistochemistry, proteomic technology is dependent on the enzymatic fragmentation of the proteins of interest and their subsequent size, chromatographic peptide separation, the mass accuracy and resolution of the mass spec-trometer, the protein database, the search algorithms, and the bioinformatics software. These vary between laboratories and can make it difficult to standardize patient results. Moreover, a variety of mass spectrometers (including an LCQ Deca XP ion trap mass spectrometer, a QSTAR-XL hybrid quadrupole-time of flight tandem mass spectrometer, a linear ion trap LTQ mass spectrometer and a LTQ-Orbitrap tandem mass spec-trometer) have been used for amyloid typing [20,26,30,35,38]. Even though all systems have been shown to produce spectra of sufficient quality and quantity to perform amyloid typing, the mass accuracy and resolution can differ drastically. These differences can be enhanced further by the chromatography

methods used up front. Protein identification is also depen-dent on the search algorithm and the cut-off scores used by the laboratory for high probability matches. The most widely used algorithms, such as Mascot, Sequest [43] and X!Tandem [44] exhibit slight differences in accuracy, sensitivity and specific-ity of mass spectra identification. This is also an area where laboratories differ [20,26,30,35,38].

Another specific issue is related to the chance that the primary sequence of the amyloidogenic proteins differs from that of the normal counterpart deposited in databases. Most peptide searching software utilize databases (such as UniProtKB/Swiss-Prot or NCBInr), which are high quality annotated and non-redundant protein sequence repositories dependent on researcher submitted protein sequences. This is an issue especially for immunoglobulin light chains, each one possessing a virtually unique primary sequence of vari-able region, making it difficult to match against the limited number of existing sequences in databases. This translates in the fact that spectra from the variable region are seldom assigned in AL patient specimens, and the most abundant spectra will match the constant regions. This is also an issue with amyloid proteins that have variants such as transthyretin, apolipoproteins and gelsolin, which sequences are not in the databases. A way to partially overcome these problems is to supplement the databases with the variant sequences of amy-loid proteins, producing better, but still not complete, amyloid protein database to search against. Well annotated specific databases of amyloidogenic light chain sequences, such as AL-Base [45], also exist and can be useful for sequence inte-gration. However, an advantage of a bottom-up proteomics approach is that once the data has been collected it is avail-able to be reprocessed as new software and protein databases become available. Additionally this allows retrospective data mining. A prime example of this benefit was observed after the report of a new amyloid type, ALect2, was published [26]. The proteomic profiles from specimens that were previously labeled as unknown amyloid type at Mayo Clinic were reana-lyzed. These profiles contained other amyloid matrix proteins such as SAP and APOE but did not have abundant spectra of any previously identified amyloid protein. Upon review of these profiles with this new information we observed the presence of the leukocyte chemotactic factor 2 protein. A large percentage of these unknown specimens were now able to be reclassified as ALect2 type.

One last issue is related to the criterion used for diagnostic interrogation of the protein lists identified in patients samples. Amyloid deposits, in fact, typically contain not only the main fibril constituent, but also variable amounts of amyloid-associ-ated proteins. Results interpretation is further complicated by the presence of contaminating serum proteins, which can be identified, generating complex protein lists. The importance of this problem becomes clear considering that all proteins responsible for systemic amyloidosis are normally found in serum at high concentrations. Care in sample preparation and the introduction of algorithms for result interpretation [35] reduce the risk of pitfalls, especially when the whole tissue is being analyzed.

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Proteomics in the evaluation of the amyloidogenic precursor

Proteomic analysis has also been applied to the study of amyloidogenic precursors in body fluids, for identifying the pathogenic species and assessing the presence of amino acid substitutions or biochemical modifications. The general strategy is the enrichment of the amyloidogenic precur-sor by on- or off-line affinity selection, followed by MS. A number of different MS approaches (determination of intact mass profiles, top-down analysis, peptide mass fingerprint-ing, MS/MS peptide sequencing etc.), allow mapping amino acid variations, assessing post-translational modifications and investigating the protein primary structure. Most of these methods could be easily adapted for use into the clini-cal chemistry laboratory. Although most efforts have been devoted to the developments of methods for analysis of serum transthyretin [4654], approaches for detailed proteomic analysis of serum monoclonal free light chains have recently been described. This could help assess the primary structure of each light chain, which at present requires bone marrow plasma cells for mRNA sequencing, and identifying signature features associated with amyloidogenicicty, to be used as future disease markers [32,55].

Conclusions and perspectives

Given their nature as protein deposition diseases, systemic amyloidoses are an ideal ground for the application of pro-teomics as a diagnostic tool. This has translated in the fact that proteomic approaches are currently used in the clinical practice, placing amyloidoses among the few examples in which this discipline has moved to the diagnostic routine. However, some practical aspects have to be underlined. Proteomics require specialized equipment and trained ana-lysts; this translates in the fact that tests are performed in specialized referral centers, where samples are to be sent. Proteomics becomes essential in the fraction of cases in which traditional analyses are not conclusive, when discrepancies between laboratory and clinical elements are observed, when concomitant elements (such as DNA mutations and mono-clonal components) are found, and in case of suspected novel amyloid types. The peculiarity of this approach also requires that clinicians be aware of the required procedures for sample acquisition and handling prior to proteomics. However, as mentioned, the most important issue is the interlaboratory standardization of the analytical approaches, with the cre-ation of quality control workflows. Establishing the proteome of a patients disease is an exciting new technology already available in the clinic today; this technology comes with its own set of complexities, many of them now instrument and informatics driven compared to older reagent driven methodologies.

Acknowledgments

We thank Prof. Giampaolo Merlini for helpful suggestions.

Declaration of interest: The authors declare no competing financial interests. F.L.s work is supported by Fondazione CARIPLO NOBEL project, Proteomic platform; EURAMY project (Communitys Sixth Framework Program); Fondazione Cariplo (N2009-2532); Ricerca Finalizzata Malattie Rare, Italian Ministry of Health, Istituto Superiore di Sanit (526D/63); Ministry of Research and University (2007AESFX2_003), and grant N. 9965 from the Associazione Italiana per la Ricerca sul Cancro Special Program Molecular Clinical Oncology.

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Proteomic typing of amyloid deposits in systemic amyloidosesIntroductionProteomics for amyloid typingProteomic identification of deposits in tissues sectionsProteomic identification of deposits in tissue biopsiesProteomic typing of amyloidoses based on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)Amyloid typing by imaging MS

Specific issues in applying proteomics to amyloid typingProteomics in the evaluation of the amyloidogenic precursorConclusions and perspectivesAcknowledgmentsReferences