Virchows Archiv
https://doi.org/10.1007/s00428-023-03591-w

BRIEF REPORT

Elucidation of the mechanism of amyloid A and transthyretin
formation using mass spectrometry‑based absolute quantification
Yukako Shintani‑Domoto1
Ryuji Ohashi1,6· Koji L. Ode2 · Seitaro Nomura3 · Hiroyuki Abe4 · Hiroki R. Ueda2,5 · Takashi Sakatani1 ·Received: 2 May 2023 / Revised: 25 June 2023 / Accepted: 27 June 2023
© The Author(s) 2023, corrected publication 2023

Abstract
Amyloidosis is triggered by the truncation of amyloid precursor proteins, causing organ damages. While previous studies
found the truncation of amyloid A (AA) and amyloid transthyretin (ATTR) occurs in C- and N-terminal, respectively, thedetailed mechanism of the fibril formation remains unclear. Liquid chromatography mass spectrometry is usually applied fora qualitative purpose, and thus quantification of tryptic peptide residue is difficult. We therefore employed a mass spectrometry-based quantification by isotope-labeled cell-free (MS-QBIC) to analyze the truncation processes in amyloid fibrillogenesis of AA and ATTR using the formalin-fixed paraffin-embedded tissues of autopsy cases. In this study, the process oftransthyretin from an 'early fibril state' consisting of full-length ATTR to a 'mature ATTR amyloid fibril' with a truncatedlow-amyloidogenic segment has been mathematically revealed. The amount of full-length ATTR was nine times higher
than in mature fibers. Large cohort studies using MS-QBIC may shed light on the clinical significance of amyloid fibrils.
Keywords Amyloidosis · Autopsy · Proteomics · HeartAbbreviations
SAA	Serum amyloid AAA	Amyloid ATTR​	Transthyretin
ATTR​	Amyloid transthyretin
FFPE	Formalin-fixed paraffin-embeddedMS-QBIC	Mass spectrometry-based quantification byisotope-labeled cell-free
LC-MS	Liquid chromatography-mass spectrometry* Yukako Shintani‑Domoto
yukakoshintani@gmail.com
1

Department of Diagnostic Pathology, Nippon
Medical School Hospital, 1‐1‐5, Sendagi, Bunkyo‐ku,Tokyo 113‐8603, Japan2

Department of Systems Pharmacology, Graduate Schoolof Medicine, The University of Tokyo, Tokyo, Japan3

Department of Cardiovascular Medicine, The Universityof Tokyo Hospital, Tokyo, Japan

4

Department of Pathology, Graduate School of Medicine, TheUniversity of Tokyo, Tokyo, Japan

5Laboratory for Synthetic Biology, RIKEN Center
for Biosystems Dynamics Research, Suita, Osaka, Japan

6Department of Integrated Diagnostic Pathology, Nippon
Medical School, Tokyo, Japan

Amyloidosis is caused by amyloid precursor proteins forming amyloid fibrils that are deposited in the extracellularmatrix, resulting in organ damage. Currently, the number ofidentified amyloid fibril proteins is 42, of which 18 proteinsappear as systemic amyloidosis [1]. Amyloid A (AA) andamyloid transthyretin (ATTR) are relatively common amyloid subtypes, but the mechanism or the structure of amyloid
fibril formation remain unclear.
We previously applied the mass spectrometry-based quantification by isotope-labeled cell-free product (MS-QBIC)
to formalin-fixed paraffin-embedded (FFPE) tissues of 30
autopsy cases with amyloidosis, including seven AA cases
and nine ATTR cases [2]. This quantification approach, theMS-QBIC method uses absolutely quantified isotope-labeled
peptides as the quantification control. (Supplemental Fig. 1)[3, 4]. Therefore, differences in ionization efficiency due todifferences in peptide sequence do not, in principle, affect thequantitative value (if the peptide to be measured is quantifiable). Note that quantification values were given within therange where the signal value changes linear to the amount ofspiked MS-QBIC peptides[2]. This approach has been feasible
and useful in classification and analysis of systemic amyloidosis. Herein, we successfully quantified the truncation processes involved in amyloid fibrillogenesis of AA and ATTR
by using MS-QBIC.

13Vol.:(0123456789)

Virchows ArchivSerum amyloid A (SAA) consists of 104 amino acids. NativeSAA, which adopts a unique four-helix bundle fold stabilized byits long C-terminal tail, exists as a hexamer. The C-terminus ofSAA is presumed to be truncated in AA amyloid fibrils; however, itremains unclear whether the C-terminus is genuinely absent or present at an under-detection level. Using MS-QBIC, amino acids (aa)26–34 and aa 91–103 of SAA peptides were quantified. Aa 26–34
was detected in all 14 AA samples, whereas the C-terminal aa
91–103 were detected in only six AA samples, illustrating that theaverage aa 26–34/aa 91–103 ratio was only 4.91% (0.95%–8.24%)
among these six samples (Fig. 1A, B). This result indicates that theC-terminus is almost absent or may be present in minimal amounts
in amyloid deposits. The length of SAA fragments detected in theamyloid fibrils varies and is dependent on the cases and/or themethods employed, shown by a previous study reporting fragments
ranging from aa 2–21 to 2–86 [5]. We recently showed that thedistribution of C-terminal tryptic peptides differed from that ofN-terminal tryptic peptides in FFPE specimens of AA patientsusing matrix-assisted laser desorption/ionization imaging mass
spectrometry [6]. These results demonstrated that the N-terminus
plays a critical role in forming AA amyloid fibrils, consistent withthe previous report [5]. A possible mechanism for this fibril formation was the activity of an endogenous protease, but the roles ofC-terminal peptides in this process require further investigation.
Transthyretin (TTR) consists of 127 amino acids. By using
cryo-electron microscopy, Schmidt et al. investigated the misfolding mechanism of TTR, i.e. that aa 36–56 is proteolyticallydegraded during the process from “early fibril state” to “matureATTR amyloid fibril” [7]. This early fibril state consists of fulllength ATTR and contains the low amyloidogenic segment at
aa 36–56 in a solvent-exposed conformation. In our study, we
have quantified two tryptic peptides; aa 22–34 and aa 36–48 of
ATTR (Fig. 2A, B) that were detected in 47 of 48 samples, withan average aa 36–48/aa 22–34 ratio of 10.26% (2.45%–26.67%),
respectively. Quantitative values of aa 36–48 indicated theamount of mature ATTR amyloid fibrils is 9 times larger thanthe early fibril state (Fig. 2C), supporting the hypothesis of TTR
misfolding, proposed by Schmidt et al. [7].
ATTR has two main fibril morphologies in amyloid
fibrils [8]. Type A fibrils are common and consist of N-terminally truncated and full-length TTR. Type B fibrils are formed
from only a few mutational variants of TTR and primarily
consist of only full-length ATTR. In our study, aa 36–48 is
significantly less common than aa 22–34, suggesting that allcases are likely to be ATTR type A. Ihse et al. reported thatpatients with ATTR fragments (type A) have a late onset ofamyloidosis and are more likely to develop cardiomyopathy,
while patients without fragments (type B) have an early onsetof amyloidosis with a higher incidence of neuropathy and alower incidence of myocardial involvement [9]. The clinicalfeature of type A and type B, classified according to the length
pattern of amyloid fibrils, is thus markedly different. Largecohort studies using our method may provide new insights
into the clinical significance of amyloid fibrils.

AB

Average of Estimated peptide concentration (n=3)(pmol/mm3)
Case Age M/F

Peptideratio

Heart

Another organ

Peptideratio

Another organ

#159

F

5.0%

0.9%

Adrenal gland

#2

78

F

5.4%

0.0%

Stomach

#371

F

4.8%

0.0%

Salivary gland

#448

M

8.2%

0.0%

Thyroid

#564

M

0.0%

0.0%

Kidney

#649

F

0.0%

0.0%

Thyroid

#775

F

5.1%

0.0%

Tongue300200100Fig. 1  Amino acid sequence and quantitative value of human Serum
amyloid A (SAA) peptides. A. In our previous study, two peptides, aa26–34 (blue and bold) and aa 91–103 (red and bold), that are boxedwere detected using absolute quantification by liquid chromatography-mass spectrometry (LC–MS) [2]. Residues 47–67 underlinedwith a blue dotted line are the C-terminal truncation site, which waspredominantly detected in a previous report [5]. B. Two specimens13

aa 26-34 (EANYIGSDK)
aa 91-103 (DPNHFRPAGLPEK)

0 0100200300were taken from each patient: cardiac tissue (cases 1–7, n = 7) andanother organ (n = 7). The bar graph shows the average of three measurements. Residues aa 26–34 were detected in all 14 AA samples,
whereas the C-terminal aa 91–103 were detected in only six AA samples, illustrating that the average aa 26–34/aa 91–103 ratio was only
4.91% (0.95%–8.24%) among these six samples

Virchows ArchivA

aa 36-48

aa 22-34

B
Average of Estimated peptide concentration (n=3)(pmol/mm3)
Case Age M/F MutantPeptideratio#965

aa 36-48 (AADDTWEPFASGK)
aa 22-34 (GSPAINVAVHVFR)

Heart

Another organ

Peptideratio

Anotherorgan

M

S50R

9.3%

26.7%

Bladder

#1075

M

V30M

11.5%

3.5%

Thyroid

#1667

M

N.A.

10.4%

5.0%

Thyroid

#1176

M

N.A.

8.2%

6.0%

Tongue

#1577

M

N.A.

9.1%

9.6%

Esophagus

#877

F

N.A.

16.6%

13.6%

Esophagus

#1484

F

N.A.

2.5%

7.6%

Stomach

#1285

M

N.A.

16.0%

#1385

M

N.A.

8.6%

(-)
(-)
600

400

20000

200

400

600

C
48
57
57
35

C34

aa 123

N 36
aa 11

1:935

NC
aa 123aa 11
22

Early fibril state

Mature ATTR amyloid fibril

Fig. 2  Amino acid sequence, quantitative value of human transthyretin (TTR) peptides and schema of folding TTR based on previously
reported data [7]. A. Transthyretin (TTR) consists of 127 amino acids.
In a previous report using the cryo-EM [7], peptides aa 11–123 weredetected (solid underlined) and 35–57 were not (dotted underlined).
Two peptides, aa 22–34 (black and bold) and aa 36–48 (red and bold),which were detected using absolute quantification by LC–MS in ourprevious study [2] are enclosed in the boxes. B. We used two specimens from each patient: cardiac tissue (n = 9) and another organ (n = 7);
however, two ATTR cases were excluded because the amount of depo-sition in organs other than the heart was too small (cases 15, 16). The
bar graph shows the average of three measurements. In our quantification study, two tryptic peptides; aa 22–34 and aa 36–48 of TTR weresuccessfully. Both fragments were detected in 47 of 48 samples, withan average aa 36–48/aa 22–34 ratio of 10.26% (2.45%–26.67%).The
original data can be found in “Supporting information (S3 Table)” inreference [2]. C. This early fibril state consists of full-length TTR andcontains the low amyloidogenic segment at aa 36–56 (including redpart). Quantitative values of aa 36–48 indicated mature ATTR amyloidfibrils are 9 times more present than the early fibril state

Our study using MS-QBIC on amyloid fibrils could mathematically capture the process by which proteolysis converts
into mature ATTR amyloid fibrils.

Funding This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant No. 17K08717 (to Y.S-D.) and20K07389 (to Y.S-D.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Supplementary Information The online version contains supplementary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 00428-0​ 23-0​ 3591-w.

Data availability All relevant data are within the paper and its Supporting Information files. The raw data of SRM analysis were deposited tothe PeptideAtlas SRM Experiment Library (PASSEL) (https://​www.​
pepti​deatl​as.​org/​PASS/​PASS0​1558).

Author contributions Conceptualization: Y. S-D., K. L. O. and T. S.;
methodology: Y. S-D., and K. L. O.; formal analysis and investigation:
Y. S-D., K. L. O., S. N., and H. A.; statistical analysis: Y. S-D.; writing— original draft preparation: Y. S-D.; writing — review and editing:
Y. S-D., K. L. O., S. N., H. A., H. R. U., T. S., and R.O.; supervision:
Y. S-D., R. O., and H. R. U.

Declarations
Informed consent The study was approved by Research Ethics Committee, Graduate School of Medicine, The University of Tokyo (No.

13Virchows Archiv10461–4-(5)). Informed consent for the use of patient specimens for.
the research was obtained from the responsible parties.
Conflict of interest H.R.U conducted a collaborative research projectwith Thermo Fisher Scientific Inc. The authors have no conflicts ofinterest to declare.
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