© Springer Science+Business Media New York 2016
Uttam Garg (ed.)Clinical Applications of Mass Spectrometry in Biomolecular AnalysisMethods in Molecular Biology137810.1007/978-1-4939-3182-8_77. Sensitive, Simple, and Robust Nano-Liquid Chromatography-Mass Spectrometry Method for Amyloid Protein Subtyping
(1)
Department of Clinical Pathology, Cleveland Clinic, Cleveland, OH, USA
(2)
Department of Laboratory Medicine, Cleveland Clinic, Cleveland, OH, USA
Abstract
Amyloidosis is a rare condition characterized by deposits of insoluble proteins in the form of β-pleated sheets. These deposits interfere with the normal structure and function of varying tissues. Thirty-one amyloid proteins have been identified, and the correct identification is critical due to the varying treatments. Immunohistochemistry, the most routine method for identification of amyloid proteins, suffers from limitations. Mass spectrometry (MS)-based methods offer better sensitivity and specificity. We describe here a sensitive, simple, and robust MS-based method for the identification of amyloid proteins. Amyloid deposits are excised from formalin-fixed tissue by laser microdissection and is put through protein extraction followed by trypsin digestion. The resulting peptides are separated by nano-liquid chromatography and analyzed by high-resolution Orbitrap mass spectrometry. The mass spectrometry data are then searched against a human protein database for identification and semi-quantification.
Key words
Amyloidosis Amyloidogenic protein β-pleated sheets Orbitrap mass spectrometry Immunohistochemistry1 Introduction
Amyloidosis is a group of rare diseases caused by extracellular protein misfolding, which generates insoluble protein deposits in the form of β-pleated sheets [1]. The fibrils generally exhibit a cross-β diffraction pattern. These fibrils bind to Congo red dye and when viewed by polarization microscopy they exhibit green, yellow, or orange birefringence color [2]. As of 2014, there are 31 known amyloidogenic protein s identified, with the most common being immunoglobulin lambda and kappa light chains, transthyretin, and serum amyloid A (SAA) which account for >90 % of the cases [3, 4]. Amyloidogenic protein s can present in various organs including but not limited to heart, liver, kidney, lung, central nervous system, skin, and cornea [3]. Amyloid deposits’ clinical presentation can be very diverse ranging from asymptomatic to multiorgan failure. Amyloid deposits can also be localized or systemic [5]. Treatments are available for many types of amyloidosis , however these treatments are type-specific ranging from high-dose chemotherapy to liver transplantation [5]. Due to the radically diverse and aggressive nature of these treatment options, accurate subtyping of amyloidosis is essential [6].
In clinical practice, amyloid identification is a two-step approach. The first step is to determine the presence of amyloid deposits. Congo red staining is considered the gold standard approach for identifying amyloid [5]. Once the presence of amyloid has been confirmed, subtyping must be performed to identify the amyloidogenic protein . The most routine method for this identification is immunohistochemistry staining of formalin-fixed and paraffin-embedded (FFPE) tissue, however this method is prone to limitations. One limitation is that wild-type antibodies may not cross-react with same protein in the amyloid deposit. A second limitation is the antibody availability. Due to these factors as many as 30 % of cases cannot be identified by immunohistochemistry [7–9]. Several mass spectrometry (MS) methods have been developed for the direct analysis of amyloid protein from FFPE tissue. MS methods have shown to have superior sensitivity and specificity to immunohistochemistry methods [10]. The following chapter describes a sensitive, simple, and robust proteomic method for amyloid subtyping that has been validated for clinical use.
2 Materials
2.1 Samples
Formalin-fixed and paraffin-embedded tissue (FFPE) is laser-microdissected and the excised tissue is transferred to 0.5 mL Eppendorf tubes. Microdissected samples are stored at −70 °C until analysis. FFPE samples are stable indefinitely at −20 and −70 °C.
2.2 Solvents and Reagents
1.
0.5 M Tris base: Weigh out 6.057 g of Tris and add to beaker containing 90 mL of clinical laboratory reagent water (CLRW). Put a mixer and stir solution. Adjust pH to 7.8 using concentrated HCl. Quantitatively transfer contents to 100 mL volumetric flask. Bring to 100 mL water. Stable for 1 year at 2–8 °C.
2.
0.1 M EDTA: Weigh out 2.9224 g of EDTA and place into a 100 mL class A volumetric flask. Bring to 100 mL with CLRW. Stable for 1 year at 2–8 °C.
3.
Digestion Buffer (10 mM Tris/1 mM EDTA/0.002 % Zwittergent 3-16, pH 7.5–8.5): Pipette 1 mL of 0.5 M Tris base into 50 mL class A volumetric flask. Pipette 0.5 mL of 0.1 M EDTA into same class A volumetric. Add 10 mg of Zwittergent 3-16 to class A volumetric flask. Bring to 50 mL with CLRW. Use HCl or KOH to pH solution between 7.8 and 8.0 if needed. Aliquot into 1.5 mL Eppendorf tubes. Stable for 5 years at −70 °C.
4.
Trypsin Buffer (50 mM Acetic Acid, pH 3): Add 30 μL of glacial acetic acid to 10 mL class A volumetric flask. Bring to 10 mL with CLRW. Aliquot into glass Teflon lined screw top vials. Discard after use.
5.
20 μg/mL trypsin solution: Add 1 mL of trypsin buffer to 20 μg of trypsin. Mix vial well. Stable for 1 year at −70 °C.
6.
2.5 % Formic Acid/5 % ACN/92.5 % CLRW: Pipette 2.5 mL of formic acid and 5 mL of acetonitrile (ACN) into 100 mL class A volumetric flask. Bring to 100 mL with CLRW. Stable for 6 months at room temperature.
7.
50 mM Dithiothreitol (DTT): Weigh out 3.8 mg of DTT into 1.5 mL Eppendorf tube. Add 0.5 mL of 2.5 % Formic Acid/5 % ACN/92.5 % CLRW and vortex. Discard this solution after use.
8.
Mobile Phase A (0.2 % Formic Acid in H2O): Add 25 mL of Fisher Optima grade H2O to mobile phase bottle. Pipette 50 μL of formic acid into bottle. Stable for 6 months at room temperature.
9.
Mobile Phase B (0.2 % Formic Acid in Acetonitrile): Add 25 mL of acetonitrile to mobile phase bottle. Pipette 50 μL of formic acid into bottle. Stable for 6 months at room temperature.
2.3 Injection Standards (Human Serum Albumin, HSA Digest)
A digested HSA solution (testmix) is prepared to test the instrument analytical performance before analyzing patient samples.
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