Veronica Valentini

Reliable typing of systemic amyloidoses through proteomic analysis of subcutaneous adipose tissue

Considering the important advances in treating specific types of systemic amyloidoses, unequivocal typing of amyloid deposits is now essential. Subcutaneous abdominal fat aspiration is the easiest, most common diagnostic procedure. We developed a novel, automated approach, based on Multidimensional Protein Identification Technology, for typing amyloidosis. Fat aspirates were obtained from patients with the most common systemic amyloidoses (ALλ, ALκ, transthyretin, and reactive amyloidosis), with Congo red score more than or equal to 3+, and nonaffected controls. Peptides from extracted and digested proteins were analyzed by Multidimensional Protein Identification Technology. On semiquantitative differential analysis (patients vs controls) of mass spectrometry data, specific proteins up-represented in patients were identified and used as deposit biomarkers. An algorithm was developed to classify patients according to type and abundance of amyloidogenic proteins in samples; in all cases, proteomic characterization was concordant with fibril identification by immunoelectron microscopy and consistent with clinical presentation. Our approach allows reliable amyloid classification using readily available fat aspirates.

A novel approach for the purification and proteomic analysis of pathogenic immunoglobulin free light chains from serum.

An excess of circulating monoclonal free immunoglobulin light chains (FLC) is common in plasma cell disorders. A subset of FLC, as amyloidogenic ones, possess intrinsic pathogenicity. Because of their complex purification, little is known on the biochemical features of serum FLC, possibly related to their pathogenic spectrum. We developed an immunopurification approach to isolate serum FLC from patients with monoclonal gammopathies, followed by proteomic characterization. Serum monoclonal FLC were detected and quantified by immunofixation and immunonephelometry. Immunoprecipitation was performed by serum incubation with agarose beads covalently linked to polyclonal anti-κ or λ FLC antibodies. Isolated FLC were analyzed by SDS-PAGE, 2D-PAGE, immunoblotting, mass spectrometry (MS). Serum FLC were immunoprecipitated from 15 patients with ALλ amyloidosis (serum λ FLC range: 98-2350mg/L), 5 with ALκ amyloidosis and 1 with κ light chain (LC) myeloma (κ FLC range: 266-2660mg/L), and 3 controls. Monoclonal FLC were the prevalent eluted species in patients. On 2D-PAGE, both λ and κ FLC originated discrete spots with multiple pI isoforms. The nature of eluted FLC and coincidence with the LC sequence from the bone marrow clone was confirmed by MS, which also detected post-translational modifications, including truncation, tryptophan oxidation, cysteinylation, peptide dimerization. Serum FLC were purified in soluble form and adequate amounts for proteomics, which allowed studying primary sequence and detecting post-translational modifications. This method is a novel instrument for studying the molecular bases of FLC pathogenicity, allowing for the first time the punctual biochemical description of the circulating forms.

Shotgun Protein Profile of Human Adipose Tissue and Its Changes in Relation to Systemic Amyloidoses

In systemic amyloidosis, accumulation of misfolded proteins as extracellular amyloid fibrils in tissues causes severe organ dysfunction, but the molecular events of tissue damage related to amyloid deposition are still largely unknown. Through the use of the MudPIT proteomic approach, comprehensive protein profiles of human amyloid-affected adipose tissue from patients and its control (non-amyloid-affected) counterpart were acquired. Label-free comparison between patients and controls made it possible to highlight differences related to the presence of amyloid, by describing up- and down-represented proteins, connected into interacting networks. In particular, extracellular matrix (ECM), protein folding, lipid metabolism, and mitochondrial functions were among the most affected structural/functional pathways. The reported results, obtained with no a priori hypotheses, represent a significant step forward in the clarification of the molecular mechanisms involved in amyloidoses at tissue level and are the premise for understanding protein misfolding diseases.

Mass spectrometry-based proteomics as a diagnostic tool when immunoelectron microscopy fails in typing amyloid deposits.

Mass spectrometry (MS)-based proteomics can directly identify amyloid deposits. We describe successful proteomic amyloid typing in fat aspirates in which immunoelectron microscopy (IEM) had not provided conclusive results. Samples with amyloid fibrils not labeled by the antibodies used in IEM were referred for 2D-PAGE-based comparative proteomics. Spots unique for patients were analyzed by matrix-assisted laser desorption/ ionization time of flight MS and database search. IEM could not characterize fibrils in 2/360 cases analyzed in 2009. In both, 2D gels displayed novel spots. Patient 1 had cardiac amyloidosis, a serum monoclonal IgMl, high l FLC, and free m heavy chains. MS allowed to assign the spots to the monoclonal l LC sequenced from bone marrow. Patient 2 had cardiac, renal, and GI amyloidosis, high k FLC, and a k band at serum immunofixation. MS and database search identified the spots as a k LC. Proteomics is a useful diagnostic complement when IEM fails in amyloid typing. Introduction: Correct typing of amyloidosis is complex and is traditionally obtained using a combination of approaches. We showed that immunoelectron microscopy (IEM), which allows the contemporaneous visualization and labeling of fibrils, is a powerful tool for typing amyloid deposits in fat and other tissues [1,2]. However, it is widely recognized that antibody-based techniques can fail in correct disease typing, at least partly due to the altered conformation of the deposited proteins and their post-translational processing [3,4]. Mass spectrometry (MS)-based proteomics was shown to be effective for reliable amyloid typing, both on fresh fat tissue samples and on formalinfixed paraffin-embedded specimens [5,6]. In particular, we reported that two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)based comparative proteomics can highlight differences in the global proteome of amyloid-affected tissues with respect to the normal counterpart, and that the identification of the patient’s novel proteins allows amyloid typing [5]. Here we describe the use of 2D-PAGE-based proteomics to successfully identify amyloid deposits in fresh fat tissue obtained by fine needle aspiration, in cases in which IEM had not provided conclusive typing. Methods: IEM was performed as previously described [1] on fat tissue specimens using polyclonal antibodies against k and l light and m heavy chains, transthyretin (TTR), apolipoprotein A-I (Apo-AI) and apolipoprotein A-II (Apo-AII), fibrinogen, lysozyme, and monoclonal antibodies against serum amyloid A (SAA) (all from Dako, Glostrup, and Denmark). Cases with visible amyloid fibrils not labeled by any of the antibodies were referred for proteomics, which was performed on fresh fat aspirates, extensively washed with isotonic saline containing protease inhibitors prior to analysis, to remove blood. Proteins were extracted from fat by tissue homogenization in lysis/isoelectrofocusing buffer and separated by 2D-PAGE as described, on large format polyacrylamide gels [5]. After gel staining and imaging, the amyloid-derived spots were located by comparison with the control 2D reference map. The novel spots, present only in patients, were excised, in-gel digested with trypsin and analyzed by matrixassisted laser desorption/ionization time of flight (MALDI-TOF) MS on a Micromass MicroMX Instrument (Waters Micromass, Milford, MA). Protein identification was performed by aligning the list of peptide ion masses in the spectra with the theoretical digest of protein sequences in NCBInr database, using Mascot (Matrix Science). In case of available bone marrow, the amino acid sequence of the full-length monoclonal free light chains of patients was deduced from the genomic one, derived from the plasma cell clone [7]. The deduced sequences were subjected to theoretical tryptic digest, to compare the peptides with those observed in the MALDI-TOF mass spectra. Western blots were performed using polyclonal antibodies against l light chains and m heavy chains (Dako). Results and discussion: IEM could detect but not characterize amyloid fibrils in two of the 374 cases (0.5% of all samples; 1.4% of the amyloid positive ones) analyzed in 2009 by this technique at the Pavia Amyloid Center (Figures 1 and 2). Patient 1. A 62-year-old man presented with peripheral neuropathy, weight loss, dysphonia, and signs of cardiac amyloidosis, both echocardiographic and biochemical (serum NT-proBNP 523 ng/l; u.r.l. 332 ng/l). Amyloid fibrils had already been detected elsewhere on the biopsy of a retroperitoneal diffuse mass incidentally found at an abdominal CT scan. 64

Circulating free light chain measurement in the diagnosis, prognostic assessment and evaluation of response of AL amyloidosis: comparison of Freelite and N latex FLC assays

Abstract Background: The measurement of circulating free light chain (FLC) is essential in the diagnosis, prognostic stratification and evaluation of response to therapy in light chain (AL) amyloidosis. For more than 10 years, this has been done with an immunonephelometric assay based on polyclonal antibodies (Freelite), and cutoffs for staging and response assessment have been validated with this method. Recently, a new assay based on monoclonal antibodies (N latex FLC) has been marketed in Europe. Methods: We evaluated and compared the clinical performance of the two assays in 426 patients with newly diagnosed AL amyloidosis. Results: We found suboptimal agreement between the two methods, with differences between values obtained with the Freelite and N latex FLC assays increasing with the concentration of clonal FLC. The diagnostic sensitivity of the Freelite (82%) and N latex FLC (84%) assays was similar, and both improved to 98% in combination with serum and urine immunofixation. The concentration of FLC measured with both methods had prognostic significance. Less pronounced decreases in FLC best predicted improved survival with the N latex FLC assay (33% vs. 50%), and there was poor concordance (84%) in discrimination of responders. Conclusions: The two assays have similar diagnostic and prognostic performance. However, they are not interchangeable, and follow-up should be done with either one. New response criteria are needed for the N latex FLC assay.

Changes in tissue proteome associated with ATTR amyloidosis: insights into pathogenesis

In Transthyretin amyloidosis (ATTR), tissue deposition of transthyretin fibrils translates into a significant subversion of the tissues proteome. We used multidimensional protein identification technology for profiling the proteome of subcutaneous adipose tissue in patients with ATTR, in comparison with controls and patients with other types of amyloidoses, to identify the global proteomic changes related specifically with this disease. The adipose tissue proteome of five ATTR patients and 11 non-affected controls was analyzed. Samples from patients with Light Chain (AL) or reactive (AA) amyloidosis were studied alongside. In all ATTR samples, mass spectrometry data showed that transthyretin was specifically up-represented, being a marker of the nature of the deposits. Tissue resident proteins, involved in key biological processes, were also found to be differently represented compared to controls. The high-throughput analysis of the proteome of amyloid affected fat, combined with bioinformatic data interpretation, is a powerful tool for identification of perturbed protein expression in ATTR amyloidosis.

Proteomic characterization of amyloid deposits in transthyretin amyloidosis associated with various mutations

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS)-based proteomics is useful for characterizing amyloid deposits. We used this approach to analyze transthyretin (TTR) in fat from patients with ATTR amyloidosis associated with various mutations. Extracted proteins were separated by 2D-PAGE. Spots uniquely present in patients were located and investigated by immunoblotting and matrix-assisted laser desorption/ionization time-of-flight MS. Ten patients were studied (4 V30M, 2 E89Q, S50R, G57R, V122I, and E92K). Deposited TTR was always visible as a main train of spots (MW *14 kDa; pI *5.4). In the S50R, G57R and E89Q variants, an additional train shifted in pI was present, containing mutated TTR. MS detected variants as mass shifts in affected peptide ions. All samples but two V30M cases showed C-terminal fragments, with similar migration across mutations. Two-dimensional PAGE-based proteomics provides very characteristic maps in ATTR amyloidosis, confirming the method as a convenient way for molecular dissection of the deposits. Introduction: Amyloidogenic transthyretin amyloidosis (ATTR) is caused by tissue deposition of transthyretin as amyloid fibrils, in association with the presence of point mutations in the coding gene. The amyloid deposits have a complex composition and have been shown to consist of a mixture of wild type and variant TTR [1,2]. Moreover, the occurrence of deposited TTR fragments in a subset of patients, whose presence has been associated to particular clinical phenotypes and pathological presentations, is a known phenomenon [3,4]. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is suitable for contemporaneously resolving hundreds of different proteins in a sample, based on molecular weight (MW) and charge properties. It thus appears as an ideal tool for a fine molecular dissection of the various and heterogeneous components of amyloid deposits, even from limited amounts of tissue. We previously showed that this technique, in combination with mass spectrometry (MS), could successfully identify and characterize amyloid proteins in fresh amyloid-affected subcutaneous abdominal fat. In particular, we reported the detailed characterization of the fat proteome in a patient with Val122Ile TTR variant [5]. These pilot proteomic results indicate an even greater complexity in the composition of ATTR amyloid deposits than previously known. Here we extend the same approach to the study of deposited TTR in fat from patients with ATTR amyloidosis associated with a broader spectrum of mutations and presenting with different clinical pictures. Methods: The presence of transthyretin mutations was assessed by genomic DNA analysis. Fresh abdominal subcutaneous fat tissue (fine needle aspirates or surgical samples), obtained during diagnostic procedures upon acquisition of informed consent, were immediately frozen at 7808C. All subsequent sample handling procedures were conducted in presence of protease inhibitors. The presence of amyloid deposits was in all cases verified by Congo red analysis and confirmed by immune electron microscopy. Heart involvement was evaluated by echocardiography and by quantification of the circulating biochemical markers of amyloid heart dysfunction [6]. Proteins were extracted from samples and subjected to 2D-PAGE as previously described [5], using 3–10 non-linear pI gradient strips for isoelectrofocusing, and 8–16% polyacrylamide gradient large format gels (Biorad, Hercules, CA). Proteins were subjected to reduction and alkylation between first and second dimension. After gel staining and imaging, proteins from amyloid deposits, uniquely present in patients, were located by comparison with the control fat 2D map. These protein spots were excised, destained, washed, and in-gel digested with trypsin, followed by analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, using a Micromass MicroMX instrument (Waters Micromass, Milford, MA). Table I. Main clinical characteristics of 10 patients with ATTR amyloidosis included in the study.

The impact of renal function on the clinical performance of FLC measurement in AL amyloidosis.

Abstract Background: The measurement of circulating free light chains (FLC) is of utmost importance in immunoglobulin light chain (AL) amyloidosis, being a fundamental part of the diagnostic workup, prognostic stratification and assessment of response to therapy. Renal failure is a common feature of AL amyloidosis and can considerably affect the concentration of FLC. Methods: We assessed the impact of renal failure on the clinical performance of the Freelite assay in 982 consecutive, newly diagnosed patients with AL amyloidosis, 822 with estimated glomerular filtration rate (eGFR) ≥30 mL/min/1.73 m2, and 160 with eGFR <30 mL/min/1.73 m2. Results: The diagnostic sensitivity of the κ/λ FLC ratio was lower for λ amyloidogenic FLC in patients with renal failure (81% vs. 60%, p<0.001) and the FLC concentration had no independent prognostic significance in patients with severe renal dysfunction. However, FLC response to chemotherapy could still discriminate patients with better outcome. Conclusions: Renal failure is a relevant interference factor when using the Freelite assay for the identification of the amyloidogenic light chain and for prognostic assessment in patients with AL amyloidosis and renal failure.

Regulated expression of amyloidogenic immunoglobulin light chains in mice

In immunoglobulin light chain (AL) amyloidosis, clinical and experimental observations converge towards a direct toxic role of the amyloidogenic light chain (aLC) precursor [1–3]. However, the molecular underpinnings of AL amyloidosis remain obscure, partly due to the paucity of preclinical models for this disease. Generation of conventional transgenic mice with constitutive overexpression of human aLCs is technically hampered by the potential toxicity of these proteins, possibly resulting in embryolethality and selection against high expressor lines [4]. To overcome these limitations, we have employed the Cre/loxP system to generate a conditional transgenic mouse allowing regulated expression of an aLC.

Identification and quantification of urinary monoclonal proteins by capillary electrophoresis in AL amyloidosis

Identification and quantification of urinary monoclonal proteins by capillary electrophoresis in AL amyloidosis Francesca Russo, Veronica Valentini, Marco Basset, Tiziana Bosoni, Paolo Milani, Giovanni Ferraro, Laura Pirolini, Andrea Foli, Francesca Lavatelli, Fabrizio Belvisi, Giorgio Consogno, Mario Nuvolone, Filomena Li Bergolis, Margherita Bozzola, Riccardo Albertini, Giovanni Palladini, and Giampaolo Merlini