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Amyloid, 2011; 18(4): 177182

Copyright 2011 Informa UK, Ltd.

ISSN 1350-6129 print/ISSN 1744-2818 online
DOI: 10.3109/13506129.2011.630762


Proteomic typing of amyloid deposits in systemic amyloidoses

Francesca Lavatelli1 & Julie A. Vrana2

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

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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 associated 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 fibrillar 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 tinctorial 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 dysfunction, translating in severe and complex clinical pictures. In
systemic amyloidoses, the clinical course, treatment and prognosis 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 deposition of misfolded monoclonal immunoglobulin light chains.
Hereditary forms (consequent to mutations in genes coding
for amyloidogenic proteins) or reactive amyloidosis (associated 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 heterogeneity, the various forms have overlapping manifestations,
which make their differentiation impossible on a clinical
basis, without the auxilium of laboratory and pathology techniques. The diagnostic workflow requires the combined use
of multiple approaches for demonstrating the presence of the

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

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

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:


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178 F. Lavatelli & J. A. Vrana

amyloidogenic precursor, detecting DNA mutations associated with familial forms and identifying the nature of the amyloid 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 immunohistochemical 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 comprehensive study of the protein constituents of a biological sample,
centered on protein analysis and identification by mass spectrometry (MS). A peculiar feature of proteomics is that proteins 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 composition of amyloid deposits and in studying the molecular
features of the amyloidogenic precursors has become immediately clear.
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 for amyloid typing, along with a brief description of the proteomic methods for assessment of the circulating amyloidogenic proteins.

Proteomics for amyloid typing

The development of MS-based methods for amyloid typing has been driven by the need for unbiased identification of the protein constituents of the deposits [813].
Immunohistochemistry and immunoelectron microscopy
have been extensively used for this purpose. However, recognized 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 paraffinembedded (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 features differentiate the various approaches, including: (1) the
tissues on which the analysis can be performed; (2) the specific 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 interpretation of results.
All the described approaches were shown to provide reliable 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 sections

Several 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 developments using either macrodissection or laser microdissection 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 proteomic profiles (ATTR, AA, ALect2, AL- and AL-). In addition to proteins involved in amyloid such as serum amyloid P
(SAP) and apolipoprotein E (APOE), the profile also identifies tissue specific proteins. As our understanding and characterization 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 referenced 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 biopsies
Proteomics 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 various 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.

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 peptide identification (peptide identifications were accepted if established
at >90% probability as specified by the Peptide Prophet algorithm)
were accepted.

A key preparative step is sample washing prior to protein

extraction, to remove blood contaminants. The average proteomic 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 differential 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 increasing the reliability of typing and eliminating the confounding
effects of blood contaminants.

Proteomic typing of amyloidoses based on twodimensional polyacrylamide gel electrophoresis

This approach has been developed for diagnostic typing of
amyloid deposits in unfixed abdominal subcutaneous fat aspirates[18,3739]. The method is based on protein separation
by 2D-PAGE, according to isoelectric point (pI) and molecular 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 strategy 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 characteristic 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

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




AL patients





Up-represented proteins
AL patients
ATTR patients





AA patients

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 patients; 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 etal, 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 proteoglycan 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

Copyright 2011 Informa UK Ltd.

180 F. Lavatelli & J. A. Vrana

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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 fragments of the deposited amyloidogenic proteins. As mentioned,
as all approaches based on the analysis of unfractionated tissue, this method is dependent on the availability of control
reference maps for each specific tissue under examination.

Amyloid typing by imaging MS

This 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 protein and peptide spatial distribution. First matrix is spotted
on the tissue and subsequently ionized in a discrete geometrical 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 chemical 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 referenced 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 instrument in amyloidoses. A first issue is related to standardization 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 spectrometer, 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 quadrupoletime of flight tandem mass spectrometer, a linear ion trap LTQ
mass spectrometer and a LTQ-Orbitrap tandem mass spectrometer) 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 dependent 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 specificity 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 variable 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 amyloid 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 integration. However, an advantage of a bottom-up proteomics
approach is that once the data has been collected it is available 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 reanalyzed. 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-associated 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 precursor 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 fingerprinting, 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 clinical 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 proteomics 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 analysts; 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 monoclonal 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 creation 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

We thank Prof. Giampaolo Merlini for helpful suggestions.
Copyright 2011 Informa UK Ltd.

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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