Article pubs.acs.org/jpr
Isobaric Quantification of Cerebrospinal Fluid Amyloid‑β Peptides in Alzheimer’s Disease: C‑Terminal Truncation Relates to Early Measures of Neurodegeneration Magnus Rogeberg,*,†,‡ Ina Selseth Almdahl,† Marianne Wettergreen,†,‡ Lars N.G. Nilsson,§ and Tormod Fladby†,∥ †
Department of Neurology, Akershus University Hospital, 1478 Lørenskog, Norway Department of Clinical Molecular Biology (EpiGen), Division of Medicine, and ∥Department of Neurology, Faculty Division, Akershus University Hospital and University of Oslo, 1478 Lørenskog, Norway § Department of Pharmacology, University of Oslo and Oslo University Hospital, 0372 Oslo, Norway ‡
S Supporting Information *
ABSTRACT: The amyloid beta (Aβ) peptide is the main constituent of the plaques characteristic of Alzheimer’s disease (AD). Measurement of Aβ1−42 in cerebrospinal fluid (CSF) is a valuable marker in AD research, where low levels indicate AD. Although the use of immunoassays measuring Aβ1−38 and Aβ1− 40 in addition to Aβ1−42 has increased, quantitative assays of other Aβ peptides remain rarely explored. We recently discovered novel Aβ peptides in CSF using antibodies recognizing the Aβ middomain region. Here we have developed a method using both Aβ N-terminal and mid-domain antibodies for immunoprecipitation in combination with isobaric labeling and liquid chromatography− tandem mass spectrometry (LC-MS/MS) for relative quantification of endogenous Aβ peptides in CSF. The developed method was used in a pilot study to produce Aβ peptide profiles from 38 CSF samples. Statistical comparison between CSF samples from 19 AD patients and 19 cognitively healthy controls revealed no significant differences at group level. A significant correlation was found between several larger C-terminally truncated Aβ peptides and protein biomarkers for neuronal damage, particularly prominent in the control group. Comparison of the isobaric quantification with immunoassays measuring Aβ1−38 or Aβ1−40 showed good correlation (r2 = 0.84 and 0.85, respectively) between the two analysis methods. The developed method could be used to assess disease-modifying therapies directed at Aβ production or degradation. KEYWORDS: Alzheimer’s disease, amyloid beta, cerebrospinal fluid, immunoprecipitation, isobaric labeling, mass spectrometry, proteomics, quantification, tau protein
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secretase, β-secretase cleavage at 10/11 and 34/35, among others.5−7 Although production of Aβ peptides has several routes, the degradation of Aβ rather than the production has been suggested to be dysregulated in sporadic AD; effort has therefore been aimed toward identifying enzymes and the mechanism behind Aβ degradation.8−10 Gaining more knowledge about the changes in Aβ peptide profiles is important as these reflect both Aβ production from AβPP through secretase activity and clearance by degradation protease activity. In addition, knowledge about the specific Aβ products and their concentration can be used for assessing toxicity and aggregation properties.11,12 While immunoassays are often used to measure a few specific Aβ peptides, mass
INTRODUCTION
Alzheimer’s disease (AD) is a neurodegenerative disease hallmarked by deposition of amyloid beta (Aβ) peptides and tau proteins in the brain. Measurement of Aβ1−42, total tau (T-tau), and phosphorylated tau (P-tau) protein in cerebrospinal fluid (CSF) is widely used as disease biomarkers.1 While the Aβ1−42 concentration reflects amyloid deposition in the brain, T-tau and P-tau serve as biomarkers for neuronal damage.2 Although Aβ-induced tau pathology is central to the current understanding of AD pathogenesis, the direct link between the two has been elusive.3 Aβ is produced from the amyloid-β precursor protein (AβPP), with full-length Aβ peptides starting at position 1 and ending at position 37 to 42 after cleavage by β- and γ- secretase at the N- and Cterminus, respectively.4 Other sites of cleavage include the nonamyloidogenic cleavage between residues 16 and 17 by α© 2015 American Chemical Society
Received: July 17, 2015 Published: October 9, 2015 4834
DOI: 10.1021/acs.jproteome.5b00668 J. Proteome Res. 2015, 14, 4834−4843
Article
Journal of Proteome Research
immunosorbent assay kits (Innotest hTau Ag, Phospho-Tau 181P, β-amyloid 1−42) obtained from Innogenetics. Immunoassay measurements of Aβ1−38 and Aβ1−40 were performed according to the manufacturers’ procedure using an MSD multi-spot assay system (Meso Scale Discovery) with 6E10 as the detection antibody. Exclusion criteria for both groups were any ongoing severe neurological, medical, or psychiatric co-morbidity or treatment with the potential to impair cognitive functioning. Exclusion criterion for the AD group was a total CSF protein concentration above 0.6 g/L (to ensure complete protein/ peptide labeling with TMT tags). The two groups were matched for age and gender. Demographics and clinical characteristics are listed in Table 1. The Regional Committee for Medical and Health Research Ethics, South East Norway, approved the study. Written informed consent was obtained from all participants.
spectrometry (MS) can be used to identify a high number of endogenous Aβ- and AβPP-derived peptides.13,14 Due to the high complexity of the CSF proteome, immunoprecipitation has often been used to reduce sample complexity prior to analysis, allowing identification of a higher number of Aβ fragments.15,16 Direct quantitation in immunoprecipitation LCMS/MS is hampered by variations from both recoveries in the precipitation steps and electrospray stability in the LC-MS/MS analysis. Using internal standards or labeling peptides is therefore common in MS-based quantification.17,18 Isobaric labeling of peptides has the advantage to allow both peptide quantification and sample multiplexing, granting the possibility for several samples to be combined and quantified in a single LC-MS/MS analysis. Due to the low protein concentration in CSF, samples can be labeled before any sample fractionation, extraction, or immunoprecipitation, and thereby, variation throughout sample preparation and LC-MS/MS analysis can be compensated.19 Reduced sample complexity after immunoprecipitation also reduces spectral interferences from coisolation of two or more peptides before fragmentation and isobaric quantification.20 To our knowledge, the combination of isobaric labeling followed by immunoprecipitation and LC-MS/MS for the quantification of Aβ peptides has not previously been reported. The current study provides a method for quantification of Aβ peptides in CSF and aims to give more insight into Aβ peptide profiles in AD through a pilot study. The developed workflow can be used in mechanistic studies of Aβ peptide production and degradation and potentially also in monitoring the effect of disease-modifying therapies.
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Table 1. Clinical Characteristicsa NC N gender (f/m) age MMSE APOEε4, n (%) Aβ42, ng/L T-tau, ng/L P-tau, ng/L
p
AD
19 10/9 65 (60−68) 29 (28−30) 4 (21%)
19 9/10 65 (60−68) 27 (23−27) 16 (84%)
0.85b
