| Metadata |
| datasetIdentifier | PASS01558 |
| datasetType | SRM |
| submitter | Koji Ode <kojiode@m.u-tokyo.ac.jp> |
| submitter_organization | The University of Tokyo |
| lab_head_full_name | Hiroki R. Ueda |
| lab_head_email | uedah-tky@umin.ac.jp |
| lab_head_organization | The University of Tokyo |
| lab_head_country | Japan |
| datasetTag | Amyloid_MSQBIC |
| datasetTitle | Mass spectrometry-based absolute quantification of amyloid proteins in pathology tissue specimens |
| publicReleaseDate | 2020-07-02 00:00:00 |
| finalizedDate | 2020-07-02 08:02:48 |
| summary | To clarify the significance of quantitative analysis of amyloid proteins in clinical practice and in research relating to systemic amyloidosis, we applied mass spectrometry–based quantification by isotope-labeled cell-free products (MS-QBIC) to formalin-fixed, paraffin-embedded (FFPE) tissues. The technique was applied to amyloid tissues collected by laser microdissection of Congo red–stained lesions of FFPE specimens. Twelve of 13 amyloid precursor proteins were successfully quantified, including serum amyloid A (SAA), transthyretin (TTR), immunoglobulin kappa light chain (IGK), immunoglobulin lambda light chain (IGL), beta-2-microglobulin (B2M), apolipoprotein (Apo) A1, Apo A4, Apo E, lysozyme, Apo A2, gelsolin, and fibrinogen alpha chain; leukocyte cell–derived chemotaxin-2 was not quantified. |
| contributors | Makiko Ogawa, Yukako Shintani-Domoto1, Yoshiki Nagashima, Koji L. Ode, Aya Sato, Yoshihiro Shimizu, Kenichi Ohashi, Michael H. A. Roehrl, Tetsuo Ushiku, Hiroki R. Ueda, Masashi Fukayama |
| publication | PMID: 32609750 |
| growth | |
| treatment | A 10 µm FFPE section of each tissue sample was stained with Congo red. Positively stained areas were dissected using the PALM MicroBeam LMD system. |
| extraction | |
| separation | |
| digestion | The samples were collected in the phase-transfer surfactant (PTS) buffer. Enzymatic digestion of amyloid samples was performed basically according to the PTS protocol. In brief, the sample was dissolved in the PTS buffer and heated at 95°C for 3 hours to reverse the formalin-cross linking. The sample was reduced with 100 mM dithiothreitol at room temperature for 30 min, and then alkylated with 1M iodoacetamide at room temperature for 30 min. Next, the sample was digested by adding 1 μg lysyl endopeptidase (Lys-C). After incubating the samples at 37°C for 3 h, 1 μg trypsin was added and further incubated at 37°C overnight. The detergents were removed by ethyl acetate/TFA solution according to the PTS method. The digested samples were then desalted by using self-prepared C18 stage tip. The recovered peptides were solubilized in 2% acetonitrile and 0.1% TFA and mixed with spike peptides prepared by MS-QBIC method. |
| acquisition | Peptide quantification was carried out by selected reaction monitoring (SRM) analysis using a TSQ Quantiva triple-stage quadrupole mass spectrometer (Thermo Fisher Scientific). The following parameters were selected: positive mode, scan width of 0.002 m/z, Q1 and Q3 resolutions of 0.7 FWHM, cycle time of 3 s, gas pressure of 1.8 torr). The spectrometer was equipped with an UltiMate 3000 RSLCnano nano-high performance liquid chromatography (HPLC) system (Thermo Fisher Scientific), and a PepMap HPLC trap column (C18, 5 µm, 100 A; Thermo Fisher Scientific) for loading samples. Analytical samples were solubilized in 2% acetonitrile and 0.1% TFA, and separated by reversed-phase chromatography using a PepMap rapid separation liquid chromatography (RSLC) EASY-Spray column (C18, 3 µm, 100 A, 75 µm x 15 cm; Thermo Fisher Scientific) using mobile phases A (0.1% formic acid/H2O) and B (0.1% formic acid and 100% acetonitrile) at a flow late 300 nL/min (4% B for 5 min, 4% to 36% B in 20 min, 36% to 95% B in 1 min, 95% B for 5 min, 95% to 4% B in 1 min and 4% B for 13 min). The elution was directly electro-sprayed into the MS. SRM transitions of the target peptides were optimized using Pinpoint software, version 1.3 (Thermo Fisher Scientific). |
| informatics | The target peptide derived from the amyloid sample was quantified by comparing the intensities of the nonlabeled target peptide in the sample with that of the stable-isotope labeled MS-QBIC peptide of known concentration. The Quan Browser of the Xcalibur data system, version 2.2 (Thermo Fisher Scientific) was used for data processing. |
| instruments | Thermo Scientific TSQ quantiva |
| species | Human |
| massModifications | static: C+57.021464, variable: K+8.014199, R+10.008269 |
