Classification of Amyloidosis by Model-Assisted Mass Spectrometry-Based Proteomics

Abstract

Amyloidosis is a rare disease caused by the misfolding and extracellular aggregation of proteins as insoluble fibrillary deposits localized either in specific organs or systemically throughout the body. The organ targeted and the disease progression and outcome is highly dependent on the specific fibril-forming protein, and its accurate identification is essential to the choice of treatment. Mass spectrometry-based proteomics has become the method of choice for the identification of the amyloidogenic protein. Regrettably, this identification relies on manual and subjective interpretation of mass spectrometry data by an expert, which is undesirable and may bias diagnosis. To circumvent this, we developed a statistical model-assisted method for the unbiased identification of amyloid-containing biopsies and amyloidosis subtyping. Based on data from mass spectrometric analysis of amyloid-containing biopsies and corresponding controls. A Boruta method applied on a random forest classifier was applied to proteomics data obtained from the mass spectrometric analysis of 75 laser dissected Congo Red positive amyloid-containing biopsies and 78 Congo Red negative biopsies to identify novel “amyloid signature” proteins that included clusterin, fibulin-1, vitronectin complement component C9 and also three collagen proteins, as well as the well-known amyloid signature proteins apolipoprotein E, apolipoprotein A4, and serum amyloid P. A SVM learning algorithm were trained on the mass spectrometry data from the analysis of the 75 amyloid-containing biopsies and 78 amyloid-negative control biopsies. The trained algorithm performed superior in the discrimination of amyloid-containing biopsies from controls, with an accuracy of 1.0 when applied to a blinded mass spectrometry validation data set of 103 prospectively collected amyloid-containing biopsies. Moreover, our method successfully classified amyloidosis patients according to the subtype in 102 out of 103 blinded cases. Collectively, our model-assisted approach identified novel amyloid-associated proteins and demonstrated the use of mass spectrometry-based data in clinical diagnostics of disease by the unbiased and reliable model-assisted classification of amyloid deposits and of the specific amyloid subtype.

Tissue Characterization in Cardiac Amyloidosis

“…LCM increases the sensitivity of the analysis by reducing the contribution of proteins from the cellular background of the tissue. Protein extraction from FFPE samples involves prolonged heating in the presence of surfactants (90 min to overnight) [ 58 , 60 , 74 , 75 , 76 ], while adipose tissue is usually sonicated for a shorter period of time (15 s to 30 min) [ 72 , 73 ]. Dialysis [ 66 ] or molecular filters [ 74 ] can be used to remove surfactants used for tissue solubilization and to exchange the buffer to one better suited for proteolytic digestion.…”

“…Dialysis [ 66 ] or molecular filters [ 74 ] can be used to remove surfactants used for tissue solubilization and to exchange the buffer to one better suited for proteolytic digestion. Disulfide bonds can be reduced and alkylated [ 74 , 75 ] to aid protein denaturation and thereby increase the accessibility of the protease to the protein backbone. Proteins are then digested using a protease, typically trypsin, at 37 °C overnight [ 75 , 76 ] or, for shorter turn-around times, for 2 h [ 65 , 74 ].…”

“…Disulfide bonds can be reduced and alkylated [ 74 , 75 ] to aid protein denaturation and thereby increase the accessibility of the protease to the protein backbone. Proteins are then digested using a protease, typically trypsin, at 37 °C overnight [ 75 , 76 ] or, for shorter turn-around times, for 2 h [ 65 , 74 ]. Interestingly, some protocols do not include protein reduction and alkylation [ 65 ] or perform the reduction after the tryptic digestion [ 74 ].…”

“…Interestingly, some protocols do not include protein reduction and alkylation [ 65 ] or perform the reduction after the tryptic digestion [ 74 ]. The peptides produced by proteolytic digestion, often referred to as tryptic peptides, are then purified using C18 solid phase extraction columns, vacuum-dried and resuspended for the chromatographic injection [ 74 , 75 ]. A summary of the workflow is shown in Figure 9 .…”

“…Tryptic peptides are analyzed by liquid chromatography coupled with mass spectrometry. The most common setup consists of a C18 chromatography coupled with the mass spectrometer by an electrospray ionization source [ 74 , 75 , 76 ], but two-dimensional chromatography has also been used to increase the number of detected peptides [ 56 , 59 , 66 ]. Most mass spectrometry methods used for amyloidosis typing are data-dependent analyses (DDA) [ 58 , 60 , 65 , 73 , 75 , 76 ].…”