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. 2023 Mar 1;34(3):505-512.
doi: 10.1021/jasms.2c00270. Epub 2023 Jan 27.

Matrix Development for the Detection of Phosphorylated Amyloid-β Peptides by MALDI-TOF-MS

Thomas Liepold  1 , Hans-Wolfgang Klafki  2 , Sathish Kumar  3 , Jochen Walter  3 , Oliver Wirths  2 , Jens Wiltfang  2 , Olaf Jahn  1   2
Affiliations

Matrix Development for the Detection of Phosphorylated Amyloid-β Peptides by MALDI-TOF-MS

Thomas Liepold et al. J Am Soc Mass Spectrom. .

Abstract

Amyloid-β (Aβ) peptides, including post-translationally modified variants thereof, are believed to play a key role in the onset and progression of Alzheimer's disease. Suggested modified Aβ species with potential disease relevance include Aβ peptides phosphorylated at serine in position eight (pSer8-Aβ) or 26 (pSer26-Aβ). However, the published studies on those Aβ peptides essentially relied on antibody-based approaches. Thus, complementary analyses by mass spectrometry, as shown for other modified Aβ variants, will be necessary not only to unambiguously verify the existence of phosphorylated Aβ species in brain samples but also to reveal their exact identity as to phosphorylation sites and potential terminal truncations. With the aim of providing a novel tool for addressing this still-unresolved issue, we developed a customized matrix formulation, referred to as TOPAC, that allows for improved detection of synthetic phosphorylated Aβ species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. When TOPAC was compared with standard matrices, we observed higher signal intensities but minimal methionine oxidation and phosphate loss for intact pSer8-Aβ(1-40) and pSer26-Aβ(1-40). Similarly, TOPAC also improved the mass spectrometric detection and sequencing of the proteolytic cleavage products pSer8-Aβ(1-16) and pSer26-Aβ(17-28). We expect that TOPAC will facilitate future efforts to detect and characterize endogenous phosphorylated Aβ species in biological samples and that it may also find its use in phospho-proteomic approaches apart from applications in the Aβ field.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Detection of intact Aβ(1–40) and pSer8-Aβ(1–40) by MALDI-TOF-MS. Mass spectra of Aβ(1–40) (left column) and pSer8-Aβ(1–40) (right column) were acquired from CHCA (A) or from THAP with different matrix additives (B). Within the THAP-based matrices, data acquisition conditions were kept constant to allow for the semiquantitative assessment of signal intensities (mean of n = 3 ± standard deviation) shown in (C). Specific signals in the mass spectra are highlighted by colored framing as follows: dark blue, nonphosphorylated Aβ without Met oxidation (-P/-O); light blue, nonphosphorylated Aβ with Met oxidation (-P/+O); red, phosphorylated Aβ without Met oxidation (+P/-O); orange, phosphorylated Aβ with Met oxidation (+P/+O). The same color code is used for the bar graphs in (C).
Figure 2
Figure 2
S/N determined from dilution series of Aβ peptides. These ratios were derived from signals for Aβ(1–40) (A) and pSer8-Aβ(1–40) (B) using THAP/DAC (open bars) or TOPAC (filled bars) as matrices. S/N for the respective amount on target (in pmol) are plotted on a logarithmic scale with standard deviations from three independent experiments. S/N = 3 (marked by stippled line) was considered as detection limit. Note that 10 fmol pSer8-Aβ(1–40) is still detectable with the TOPAC matrix.
Figure 3
Figure 3
Sequencing of phosphorylated Aβ cleavage products by MALDI-TOF/TOF-MS/MS. Fragment ion mass spectra of pSer8-Aβ(1–16) (left column) and pSer8-Aβ(17–28) (right column) were acquired from THAP/DAC (upper row) and TOPAC (lower row). Only b- and y-ion series are labeled for the sake of clarity. For pSer8-Aβ(1–16), phosphorylation was clearly assigned to Ser-8 (marked in red) on the basis of conclusive N- and C-terminal ion series. The high abundance of the N-terminal b7-ion was in agreement with the preferential cleavage of peptide bonds C-terminal of Asp under the conditions of mass spectrometric peptide sequencing. Similarly, for pSer26-Aβ(17–28), phosphorylation was clearly assigned to Ser-26 (marked in red). Here, the y-ion series was more abundant in agreement with charge localization at the C-terminal Lys residue and was accompanied by a y-ion series showing loss of phosphoric acid (−98 Da, only annotated and marked by -P in the fragment ion mass spectrum from TOPAC matrix). Additional fragment ions detected exclusively from TOPAC matrix are marked in magenta. The images of the MALDI target plate spots (insets) show a virtually identical crystallization behavior of both matrices. This indicates that the matrix additives of the TOPAC formulation apparently did not alter the crystallization behavior of THAP/DAC, which may be of relevance when automated data acquisition is desired.
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