Mass spectrometric analysis of accumulated TDP-43 in amyotrophic lateral sclerosis brains

Abstract

TDP-43 is the major disease-associated protein involved in the pathogenesis and progression of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions linked to TDP-43 pathology (FTLD-TDP). Abnormal phosphorylation, truncation and cytoplasmic mis-localization are known to be the characteristics for the aggregated forms of TDP-43, and gain of toxic abnormal TDP-43 or loss of function of physiological TDP-43 have been suggested as the cause of neurodegeneration. However, most of the post-translational modifications or truncation sites in the abnormal TDP-43 in brains of patients remain to be identified by protein chemical analysis. In this study, we carried out a highly sensitive liquid chromatography-mass spectrometry analysis of Sarkosyl-insoluble pathological TDP-43 from brains of ALS patients and identified several novel phosphorylation sites, deamidation sites, and cleavage sites. Almost all modifications were localized in the Gly-rich C-terminal half. Most of the cleavage sites identified in this study are novel and are located in N-terminal half, suggesting that these sites may be more accessible to proteolytic enzymes. The data obtained in this study provide a foundation for the molecular mechanisms of TDP-43 aggregation and ALS pathogenesis.

Figure 1. SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting of Sarkosyl-insoluble fraction from ALS case 1 brain and ALS case 2 brain.

(A) A phosphorylation-independent anti-TDP-43 detected normal TDP-43 of 43 kD in the fractions of both ALS and control brains, but also detected abnormal TDP-43 bands, including full-length phosphorylated TDP-43 of 45 kD, ~25 kD fragments and smears in ALS brains. (B) TDP-43-positive bands were excised as indicated.

Figure 2. MS/MS identification of phosphorylated peptides and cleavage site peptides.

Representative peptides were shown. (A) Chymotriptic peptide, 398-NGGFGSSM-405 in case 1. The 6th Ser residue was phosphorylated. (B)Triptic peptide, 56-LVEGILHAPDAGWGNLVYVVNYPK-79 in case 1. (C) Tryptic peptide, 215- GDVMDVFIPKPFR-227 in case 1. Trypsin can not cleave N-terminal Tyr214-Gly215 site. Therefore this site is intrinsically cleaved site. (D) Chymotryptic peptide, 56-LVEGILHAPDAGW-68 in case 2. Chymotrypsin can not cleave N-terminal Arg55-Leu56 site. Therefore this site is intrinsically cleaved site.

Figure 3

Identification of modification sites in TDP-43 from ALS case 1 (A) and case 2 (B) by LC-MS/MS analysis. Identified peptides from trypsin digestion (light green) and from chymotrypsin digestion (dark green) are shown. p indicates phosphorylation site. o indicates oxidation site. * indicates deamidation site. Blue and pink arrows indicate N-terminal and C-terminal cleavage sites, respectively.

Figure 4. Schematic representation of N-terminal and C-terminal cleavage sites on TDP-43.

Cleavage sites (amino acid numbering of TDP-43) in gel fractions and antibodies recognition sites were shown.