Project description:Multiple Affinity Removal Spin Cartridge Human 14 kit (Agilent) was used to remove 14 major proteins from plasma. Samples were denatured with an equivalent volume of trifluoroethanol and reduced with dithiothreitol. Free cysteine residues were alkylated with iodoacetamide. Samples were incubated with trypsin and desalted with C18 ZipTip. Desalted samples were rehydrated in 0.1% formic acid (FA) and were analyzed by LC-MS using a nanoLC Eksigent 400 system (Eksigent, AB Sciex), coupled online to an TripleTOF6600 mass spectrometer (AB Sciex). Peptide separation was performed using liquid chromatography with a trap and elution conﬁguration using a nano trap column (350 μm × 0.5 mm, 3 μm, 120 Å, AB Sciex) and a nano ChromXP C18 reverse phase column (75 μm × 15 cm, 3 μm, 120 Å, AB Sciex) at 300 nl/min with a 90 min linear gradient of 8-30% acetonitrile in 0.1% FA, and then, with a 10 min linear gradient of 30% to 40% acetonitrile in 0.1% FA. The mass spectrometer was operated in information-dependent acquisition (IDA) mode, scanning full spectra (400–1500 m/z) for 250 ms, followed by up to 30 MS/MS scans (100–1800 m/z for 50 ms each), for a cycle time of 1.8 s. Candidate ions with a charge state between +2 and + 5 and counts above a minimum threshold of 125 counts per second were isolated for fragmentation, and one MS/MS spectrum was collected before adding those ions to the exclusion list for 12 s. Rolling collision energy was used with a collision energy spread of 15. The mass spectrometer was operated using the Analyst TF 1.7.1 software program (AB Sciex). For data dependent acquisition (DDA, SWATH acquisition), the parameters were set as follows: 100 ms TOF MS scan, followed by 200 variable SWATH windows each at 50 ms accumulation time for m/z 400–1250. MS/MS SWATH scans were set at 5 Da window overlapping by 1 Da for m/z 400–1250 and varied on each side of the mass range. The total cycle time was 9.6 s. A rolling collision energy (CE) parameters script was used to automatically control the CE. Acquired spectra were searched against the UniProt reviewed database using the Paragon algorithm embedded in the ProteinPilot 5.0.1 software program (AB Sciex), with the following search parameters: (i) sample type: identiﬁcation, (ii) Cys alkylation: iodoacetamide, (iii) digestion: trypsin, (iv) instrument: TripleTOF 6600, (v) species: Homo sapiens, (vi) ID focus: biological modiﬁcations, (vii) detected protein threshold: > 0.05 (10% conﬁdence). The detected protein threshold was set to the minimum level to enhance the number of wrong answers to enable the curve ﬁtting by an independent FDR analysis. This was carried out by the target-decoy approach provided with the ProteinPilot software program, which was used to assess the quality of the identiﬁcations. Positive identiﬁcations were considered when identiﬁed proteins and peptides reached a 1% local FDR. The resulting group file was loaded into Peakview (v2.2.0, AB Sciex) and peaks from SWATH runs were extracted with a peptide confidence threshold of 99% and a false discovery rate <1%. The SWATH ﬁles were then exported to the MarkerView software program (version 1.3.0.1; AB Sciex) and the peak areas of individual peptides were normalized to the sum of the peak areas of all detected peptides.

Project description:To date, most diagnostic imaging comparisons between amyloid labelling ligands and other imaging modalities have been between the use of amyloid labelling ligand (11)C Pittsburgh Compound B (PiB) and FDG-PET. Our objectives were to compare cognitive performance and diagnostic group-wise discrimination between cognitively normal, amnestic mild cognitive impairment (MCI) and Alzheimer's disease subjects with MRI-based measures of hippocampal volume and PiB retention, and secondly to evaluate the topographic distribution of PiB retention and grey matter loss using 3D voxel-wise methods. Twenty cognitively normal, 17 amnestic MCI and 8 probable Alzheimer's disease subjects were imaged with both MRI and PiB. PiB retention was quantified as the ratio of uptake in cortical to cerebellar regions of interest (ROIs) 40-60 min post-injection. A global cortical PiB retention summary measure was derived from six cortical ROIs. Statistical parametric mapping (SPM) and voxel-based morphometry (VBM) were used to evaluate PiB retention and grey matter loss on a 3D voxel-wise basis. Alzheimer's disease subjects had high global cortical PiB retention and low hippocampal volume; most cognitively normal subjects had low PiB retention and high hippocampal volume; and on average amnestic MCI subjects were intermediate on both PiB and hippocampal volume. A target-to-cerebellar ratio of 1.5 was used to designate subjects with high or low PiB cortical retention. All Alzheimer's disease subjects fell above this ratio, as did 6 out of 20 cognitively normal subjects and 9 out of 17 MCI subjects, indicating bi-modal PiB retention in the latter two groups. Interestingly, we found no consistent differences in learning and memory performance between high versus low PiB cognitively normal or amnestic MCI subjects. The SPM/VBM voxel-wise comparisons of Alzheimer's disease versus cognitively normal subjects provided complementary information in that clear and meaningful similarities and differences in topographical distribution of amyloid deposition and grey matter loss were shown. The frontal lobes had high PiB retention with little grey matter loss, anteromedial temporal areas had low PiB retention with significant grey matter loss, whereas lateral temporoparietal association cortex displayed both significant PiB retention and grey matter loss. A voxel-wise SPM conjunction analysis revealed that subjects with high PiB retention shared a common PiB retention topographical pattern regardless of clinical category, and this matched that of amyloid plaque distribution from autopsy studies of Alzheimer's disease. Both global cortical PiB retention and hippocampal volumes demonstrated significant correlation in the expected direction with cognitive testing performance; however, correlations were stronger with MRI than PiB. Pair-wise inter-group diagnostic separation was significant for all group-wise pairs for both PiB and hippocampal volume with the exception of the comparison of cognitively normal versus amnestic MCI, which was not significant for PiB. PiB and MRI provided complementary information such that clinical diagnostic classification using both methods was superior to using either in isolation.

Dataset Information

Plasma A? and PET PiB binding are inversely related in mild cognitive impairment.

Objective

Methods

Results

Conclusions

Publications

Plasma Aβ and PET PiB binding are inversely related in mild cognitive impairment.

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