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Briefings in Functional Genomics and Proteomics Advance Access originally published online on June 20, 2007
Briefings in Functional Genomics and Proteomics 2007 6(2):149-158; doi:10.1093/bfgp/elm010
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Quantitative analysis of amyloid-ß peptides in cerebrospinal fluid using immunoprecipitation and MALDI-Tof mass spectrometry

Valentina Gelfanova, Richard E. Higgs, Robert A. Dean, David M. Holtzman, Martin R. Farlow, Eric R. Siemers, Amechand Boodhoo, Yue-Wei Qian, Xiaohua He, Zhaoyan Jin, Deborah L. Fisher, Karen L. Cox and John E. Hale

Corresponding author. John E. Hale, Lilly Research Laboratories, P.O. Box 708, Greenfield, IN 46140, USA. E-mail: hale_john_E{at}lilly.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Immunoprecipitation (IP) combined with matrix-assisted laser desorption ionization (MALDI) time of flight (Tof) mass spectrometry has been used to develop quantitative assays for amyloid-ß (Aß) peptides in cerebrospinal fluid (CSF). Inclusion of 15N labelled standard peptides allows for absolute quantification of multiple Aß isoforms in individual samples. Characterization of variability associated with all steps of the assay indicated that the IP step is the single largest contributor to overall variability. Optimization of the assay resulted in overall coefficient of variation ≤8% with high agreement to an Aß1-40 and Aß1-42 ELISA assay. Application of the MALDI-Tof assay to CSF obtained from healthy volunteers and Alzheimer's disease patients indicated statistically significant 43% lower levels of Aß1-42 in the AD group (P = 0.0025).

Keywords: Alzheimers disease, amyloid-ß peptide, MALDI-Tof mass spectrometry, multiplexed analysis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Amyloid-ß (Aß) peptide is the major component of senile plaques deposited in the brains of individuals with Alzheimer's disease (AD) [1]. Aß is produced from the amyloid precursor protein (APP) through multiple proteolytic processing steps. APP is a trans-membrane protein which can be cleaved in the extracellular domain by ß-secretase [2–4]. Another protease known as {gamma}-secretase can cleave the APP in the segment of the protein that is inserted in the lipid bilayer of the cellular membrane [5]. The specificity of {gamma}-secretase is not absolute and different forms of the Aß peptide are typically seen in brain tissue and cerebrospinal fluid (CSF) [6]. The two best known forms of the Aß peptide are known as Aß1-40 and Aß1-42 depending on the number of amino acids in the peptide. Other forms of the peptide have also been observed in brain tissue which are either N- or C-terminal truncations of the peptide [7]. Amyoid plaque in brain parenchyma is a pathognomonic feature of AD. Accordingly, one predominant theory claims that increased amounts of Aß in the parenchyma mediates the neurodegenerative processes giving rise to the full spectrum of clinical findings of AD. Whether Aß monomers, oligomers or amyloid plaque is the primary toxic species is not known. For this reason, some therapeutic strategies are targeted at inhibition of ß- and {gamma}-secretases to prevent formation of Aß while others are focused on reducing Aß peptides through immunological methods [1]. For all therapies designed to reduce production of the Aß peptide, the availability of analytically sensitive and selective assays to quantify the levels of the various forms of the Aß peptide is important. ELISA assays have been developed for Aß peptides and can measure total amounts of the peptide or specific versions (i.e. 1–40 or 1–42). These assays require pairs of antibodies, at least one of which has specificity for the region of interest. Monitoring multiple forms of the peptide requires running multiple assays. Interference can arise if the antibodies do not have absolute specificity or if different forms of the Aß peptide cross-react with the antisera. Mass spectrometry (MS) has been used to characterize and profile Aß peptides [7, 8]. Matrix-assisted laser desorption ionization (MALDI) time of flight (Tof) MS is capable of resolving multiple forms of the Aß peptide based on mass. Coupling this analytical technology with immunoprecititation techniques allows the isolation and characterization of Aß from complex biological samples like plasma or CSF [9]. MALDI-Tof-MS has not widely been used to quantify peptides or proteins. Reasons for this include variability in the ionization potential for different peptides and heterogeneity in the matrix/peptide deposition. Some groups have recently begun using MALDI-Tof for quantification of proteins and have controlled for variability through inclusion of appropriate internal control proteins [10–12]. Isotopically labelled peptides have been used to quantify Lys-C digests of Aß peptide extracted from brain tissue using MALDI-MS [13]. Peptides enriched in 13C or 15N may be resolved from peptides of the same sequence by mass. These peptides are chemically identical and will behave similarly biologically and analytically. Thus, addition of 15N enriched forms of Aß peptides to biological fluids enables one to use these as internal standards in subsequent isolation and quantification steps. We report here the development of quantitative immunoprecipitation (IP)–MALDI-MS assays that can simultaneously measure levels of multiple different forms of the Aß peptide and also provide a profile of the different Aß isoforms in CSF. Individual Aß forms quantified with this assay have been correlated to values determined by an ELISA assay with high levels of correspondence.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
CSF was obtained from healthy volunteers (HV) and subjects diagnosed with AD. Samples were collected as part of studies approved by the clinical study site-specific Ethical Review Boards of Washington University (St Louis) and Indiana University. All study participants provided informed consent and underwent evaluation of medical history, physical and neurological examinations, laboratory tests and neuropsychological assessment using the Mini-Mental State Exam (MMSE) [14]. Subjects with AD also underwent cognitive testing with the Alzheimer's Disease Assessment Scale—Cognition (ADAS-cog) [15]. None of the HVs had any signs or symptoms of cognitive decline or neurological disease. All of the AD patients met the criteria for probable AD using National Institute of Neurological and Communicative Disorders/Alzheimer's Disease and Related Disorders Association (NINCDS/ADRDA) criteria [16]. CSF samples were obtained between 12:00 PM and 5:00 PM. A total of 25 ml of CSF were taken in three fractions; the CSF used in this study was taken from fraction containing millilitres 9–20. Pooled human CSF was purchased from Bioreclamation Inc. (Hicksville, NY, USA).

Uniformly 15N labelled human Aß1-38 was produced in Escherichia coli, labelled Aß1-40, and Aß1-42 were obtained from R-peptide (Atlanta, GA, USA), and human Aß1-40 was purchased from Biosource International (Camarillo, CA, USA). Dynabeads M-280 Streptavidin were purchased from Dynal (Lake Success, NY, USA). Isopropanol and formic acid were obtained from Sigma (St Louis, MO, USA). MassPREP MALDI Matrix, CHCA, was purchased from Waters (Milford, MA, USA). Saturated matrix solution was prepared by dissolving CHCA in 50% isopropanol and 5% formic acid in water.

Immunoprecipitation
CSF samples (1 ml) were spiked with internal standard(s) Aß peptides (15NAß1-38, 15NAß1-40 and/or 15NAß1-42). Biotinylated anti-Aß antibody with specificity to the mid-region of the peptide (266.2) [17] was added to a final concentration of 5 µg/ml. After incubation overnight at 4°C, biotinylated antibody was captured with streptavidin-coupled magnetic beads (3 h, RT). The beads were isolated with a magnet and the supernatant was removed. Beads were washed 3x with phosphate-buffered saline and twice with 100 mM ammonium bicarbonate buffer, pH 7.8. Bound peptides were eluted from the beads in 10% formic acid, spotted onto MALDI targets with an equal volume of saturated {alpha}-cyano-4-hydroxycinnamic acid solution added.

MALDI-mass spectrometry
All spectra were acquired with a 4700 MALDI-Tof-Tof mass spectrometer (PerSeptive Biosystems, Framingham, MA, USA) in the linear delayed extraction mode. Spectra were acquired in 12 separate areas of each spot with 200 laser shots per acquisition and all acquisitions were averaged for each spot.

Data analysis
The following criteria were used to assign the Aß peptides: (i) the measured molecular mass of the peak matched the calculated molecular mass of Aß peptide based on the human Aß amino acid sequence and (ii) the tentative matched Aß peptide contained the epitope site for the 266.2 antibody (amino acids 13–28). Software for data analyses post-acquisition from the MALDI-Tof mass spectrometer was written in-house using the R statistical computing environment [18]. The inputs to this internally developed R procedure, maldi_quant, include a list of peptide mass-to-charge ratio (m/z) values to quantify and a spectrum or a directory of spectra. Normal processing of the mass spectra included smoothing of the spectrum using a super smoother [19], local linear baseline subtraction and nonlinear regression peak fitting with a Lorentzian peak shape for quantification. Nonlinear regression was carried out using Gauss–Newton optimization as implemented in the R function nls [20]. Initial estimates of the Lorentzian peak parameters, location, scale and area were derived from the spectrum in a region around a local maximum. Calibration curves from spiked recovery experiments were fit using weighted (variance-modelled as a power of the mean) nonlinear regression with the R function gnls contained in the nlme R package [21]. Weighting was included in the calibration curve estimation due to the observed relationship of variability increasing with larger signal intensity. Assay working ranges were estimated from spiked recovery experiments by the range of validation dilutions (dilutions not used to fit the calibration curve) for which the total relative error is <30%.

ELISA for Aß quantification
CSF samples were analysed for Aß using a modified sandwich ELISA specific for human Aß1-40 or Aß1-42 [22]. To capture the different species of full-length Aß peptides, C-terminal domain monoclonal antibodies specific for either Aß1-40 (clone 2G3) or Aß1-42 (clone 21F12) were used. All assays used a biotinylated N-terminal domain monoclonal antibody specific for an intact N-terminus (amino acids 1–5) of human Aß (3D6) as a reporting antibody, followed by streptavidin-poly-horseradish peroxidase-20 (Amersham Biosciences, Piscataway, NJ, USA). TMB substrate (Pierce, Rockford, IL, USA) was used for colorimetric detection and analysed on a SpectroMax (Molecular Devices, Sunnyvale, CA, USA) microtitre plate reader. Aß1-40 and Aß1-42 standard curves were generated from Aß peptide obtained from Bachem California Inc. (Torrance, CA, USA). All ELISA samples were diluted in a final buffer of 0.1 M HEPES, 0.1 M NaCl, 0.1% human serum albumin (InstitutoGrifols), 0.1% Tween 20, pH 7.4.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Immunoprecipitation
Addition of internal standard peptides to CSF prior to addition of the anti-Aß antibody controls for any potential losses of peptide during the incubation and isolation steps. A representative spectrum of peptides eluted from the streptavidin-coated magnetic beads of an IP from 1 ml of CSF is shown in Figure 1. Peptides with masses corresponding to Aß1-33, Aß1-34, Aß1-37, Aß1-38, Aß1-39, Aß1-40 and Aß1-42 are clearly visible. Spiked internal standard peptides for Aß1-40 and Aß1-42 are also visible. No truncated forms of the labelled peptides are present, indicating that the C-terminal truncation of Aß did not occur during the incubation steps, and therefore they likely arise from in vivo processing of the peptide. IPs were conducted on 0.5 and 0.25 ml volumes of CSF and all of the listed peptides were visible (data not shown). The signal-to-noise ratio decreased, however, with these smaller volumes, decreasing the sensitivity of the assay. For this reason, 1 ml of CSF was used for all of the subsequent assays.


Figure 1
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  		Figure 1: Representative MALDI-Tof-MS of Aß peptides immunoprecipitated from CSF. * Peak present in negative control IPs.

 
IP–MALDI-Tof precision (repeatability)
To estimate the relative sources of variability of the analytical procedure, 11 1 ml aliquots of pooled CSF sample were spiked with 9 ng/ml 15N Aß1-40 and 5 µg/ml of 266.2 antibody and processed as described in the Materials and Methods section. Each eluted sample was spotted four times onto two different MALDI target plates. The response was expressed as the ratio of Aß peptides to 15N Aß1-40. A variance components analysis indicates that the reproducibility of the method is generally good, with total CV ≤8% for all Aß peptides reported, and with the majority of the total variability attributed to the IP assay step (Table 1).


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  		Table 1: Variance components analysis results from an 11 IP, 2 plates per IP, and 4 spots per plate reproducibility study

 
MALDI-Tof linearity and range (spiked recovery)
To test the linear response of the MALDI and estimate the working range of the instrument, a series of dilutions of synthetic Aß1-40 were spotted from 0.018 to 2.5 ng/spot onto three different MALDI target plates (4 spots/plate for each dilution). Each dilution contained a constant amount of 15N labelled Aß1-40 (0.125 ng/spot) and the response was expressed as the ratio of unlabelled to labeled Aß1-40. A plot of the response from one plate is shown in Figure 2. Back-calculating the concentrations of the validation samples using a weighted four-parameter logistic calibration curve estimated from the calibration samples on each plate indicated that the limit of quantification and the limit of detection of this assay was 0.08 ng/spot with a working range of at least ~20-fold (Figure 2).


Figure 2
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  		Figure 2: (A) Spiked recovery results for Aß1-40: 15N Aß1-40 ratio for one of three plates analysed in the experiment. Calibration dilutions denoted by open circles and validation samples denoted by Xs (4 spots per dilution). Weighted (variance modelled as a power of the mean) four-parameter logistic fit to the calibration dilutions shown. (B) Bias, precision and total error estimated from the between- and within-plate spiked recover validation samples. Seven dilutions used to estimate the parameters of a four-parameter logistic standard curve. Validation sample concentrations equally spaced between calibration sample dilutions. Dilutions <0.059 ng/spot were not detectable on the MALDI-Tof. Total error profile indicates that the MALDI is quantitative for the entire range of validation dilutions tested (~20-fold working range).

 
IP–MALDI-Tof linearity and range (spiked recovery)
To estimate the working range of the IP–MALDI procedure, 12 serial dilutions from a stock standard solution of 15N Aß1-38 were made in PBS/1%BSA and added to the pooled CSF aliquots (three aliquots/dilution) to obtain CSF samples with 15N Aß1-38 peptide levels from 50 ng/ml to 24 pg/ml. Samples also received 9 ng/ml of 15N Aß1-40 and 2 ng/ml of 15N Aß1-42 peptides and were subjected to the IP–MALDI procedure. Eluted peptides were spotted onto three MALDI plates (one aliquot of each dilutions/plate). The response was expressed as the ratio of 15N Aß1-38 to 15N Aß1-40 peptide. A plot of the response from one plate is shown in Figure 3. Back-calculating the concentrations of the validation samples using a weighted four-parameter logistic calibration curve estimated from the calibration samples on each plate indicates that the limit of quantification and the limit of detection of this assay is ~0.3 ng/ml with a working range of ~62-fold (Figure 3). Similar results and working ranges were estimated using 15N Aß1-42 and the endogenous Aß1-38 as the normalizing reference (data not shown).


Figure 3
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  		Figure 3: (A)Spiked recovery results for 15N Aß1-38: 15N Aß1-40 ratio for one of three plates analysed in the experiment. Calibration dilutions denoted by open circles and validation samples denoted by Xs (4 spots per dilution). Weighted (variance modelled as a power of the mean) four-parameter logistic fit to the calibration dilutions shown. (B) Bias, precision and total error estimated from the between- and within-plate spiked recover validation samples. Seven dilutions used to estimate the parameters of a weighted four-parameter logistic calibration curve. Validation sample concentrations equally spaced between calibration sample dilutions. Total error profile indicates that the IP–MALDI assay is quantitative from ~0.4 ng/ml to at least 25 ng/ml (62-fold working range).

 
IP–MALDI-Tof specificity
To demonstrate assay specificity, ratios of Aß peptides to the 15N Aß1-40 peptide were calculated for the spike-recovery experiment described above. Normalized responses for four Aß isoforms indicated no substantial effect from the titration of 15N Aß1-38 in the spiked recovery experiment (Figure 4).


Figure 4
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  		Figure 4: Normalized responses to human 15N Aß1-40 as 15N Aß1-38 is titrated for Aß1-37 (A), Aß1-38 (B), Aß1-40 (C) and Aß1-42 (D). Plotting scale is one-tenth the range of the normalized response of the 15N Aß1-38 response in Figure 3.

 
Analysis of clinical samples
CSF from 14 HVs and 13 subjects diagnosed with AD was spiked with 15N labelled Aß 1-40 and Aß1-42 and then subjected to the IP–MALDI procedure. Aliquots of the same samples were assayed by ELISA for Aß1-40. The mean values for Aß1-33 through Aß1-42 were determined after normalization to the labelled Aß1-40 standard (which was spiked at 9 ng/ml). There were no statistical differences in the group means of any of the Aß peptides from 1-33 to 1-40 (Figure 5). There did appear to be a trend with the AD group having lower levels of peptides with this difference increasing with the length of the peptide. Aß1-42 showed a significantly lower level in the AD group compared to the HV group (P = 0.0025, 43% reduction in AD group mean relative to HV group mean). This is consistent with previously published measurements of Aß1-42 in AD subjects [23] and with previous analysis of aliquots of these same samples by ELISA for Aß1-42 (data not shown). The detected Aß peptides were generally correlated with each other across subjects, with Aß1-42 showing the lowest overall correlation to the other Aß peptides and with this correlation decreasing as the length of the peptides decreases (Figure 6). Values obtained for Aß1-40 and Aß1-42 by MALDI analysis were plotted against values obtained for these same samples by ELISA (Figure 7A and 7B). There was a very good correlation between the values obtained by both assays for Aß1-40 (r = 0.95) which was more highly correlated than two ELISAs run on different aliquots of the same samples (r = 0.89, data not shown). The correlation between both assays for Aß1-42 was also very good (r = 0.88) which was equivalent to the correlation between two different ELISAs run on the same samples (r = 0.89, data not shown).


Figure 5
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  		Figure 5: AD versus HV for Aß1-33 through Aß1-42. Modelled response is log10 of the peptide area to the labelled internal standard (15N Aß1-40 for Aß1-33 through Aß1-40 and 15N Aß1-42 for Aß1-42). Each peptide was scaled such that the control group had a mean of 0. Only Aß1-42 was found to be significantly altered in the AD subjects (P = 0.0025, 43% reduction in AD versus HV group means).

 

Figure 6
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  		Figure 6: Correlation matrix of seven different Aß peptides. Values plotted are log10 of the Aß peptide area relative to the labelled internal standard (15N Aß1-40 for Aß1-33 through Aß1-40 and 15N Aß1-42 for Aß1-42). Upper matrix displays the Pearson correlation coefficient with font size proportional to the value. Aß1-42 is overall the least correlated with the other peptides with the correlation decreasing as peptide length decreases.

 

Figure 7
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  		Figure 7: Aß ELISA values versus IP–MALDI values (AD = triangles, HV = circles) for Aß1-40 (A) and Aß1-42 (B). Pearson's correlation coefficient equals 0.95 for Aß1-40 and equals 0.88 for Aß1-42. IP–MALDI value is the Aß peak area divided by the peak area for the corresponding 15N labelled standard.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Therapeutic strategies aimed at reducing the production or increasing clearance of Aß are predicated on the Amyloid hypothesis of AD [1]. This hypothesis holds that the neurodegeneration and cognitive decline associated with AD is primarily a consequence of altered production or clearance of Aß. Measurement of Aß in CSF is a potential biomarker to monitor the pharmacological activity of potential therapeutic interventions. ELISA assays have been developed and routinely used for this purpose [23]. Characterization of Aß in CSF and in brain tissue by MS has revealed that Aß exists in multiple forms besides the most commonly studied 1-40 and 1-42 forms [7, 24]. Some truncated forms of Aß have been proposed to be more fibrillogenic and toxic than Aß1-42 [25, 26]. It has been proposed that non-steroidal anti-inflamatory drug (NSAID) compounds can alter the specificity of {gamma}-secretase, increasing the production of Aß1-38 while decreasing the production of Aß1-42 [27]. Differences in the propensity of these forms of Aß to aggregate could alter the rate of plaque deposition. Monitoring multiple forms of Aß is a necessary tool for exploring this hypothesis. Using ELISA assays to accomplish this requires development of multiple assays. Antibodies specific for the different forms need to be produced and measurement requires running multiple assays. In addition, N-terminal truncations have been seen which would necessitate specific antibodies to be developed at both the ends of the Aß molecule [7]. MS is readily capable of resolving the various different forms of Aß based on mass and can resolve other chemical variants of Aß such as oxidized forms. While MS has been used to identify and profile Aß, it has the potential to quantify multiple forms of Aß from a single sample. We have chosen MALDI-Tof-MS for this purpose. MALDI-Tof-MS has rarely been used quantitatively [8, 10–13] and it has been assumed by some that the technique is too variable to be used for this purpose. We minimized the variability contributed by heterogeneity in the sample-matrix spot by appropriate sampling of the spot with the laser and averaging multiple spectra acquired from different regions within the spot. It would not be appropriate to infer quantitative information for different forms of Aß by simple comparison of peak heights or areas. This is due to the fact that peptides with different amino acid sequences may have different ionization potentials. Normalizing these peaks to 15N labelled standards can control for these differences. These standards can also serve as controls to minimize the impact of pre-analytical variability attributable to sample handling and processing steps. The overall performance of the quantitative MALDI-Tof assay reported here was very good with coefficients of variation generally <10% and the majority of the total variability attributed to the immonoprecipitation assay step. Within-subject biological variability with repeated sampling of CSF was not evaluated in this study, but should be evaluated in future investigations. The assay correlated very well with an ELISA assay for Aß1-40, and performed as expected when applied to clinical samples. As reported in numerous other studies,Aß1-42 demonstrated significantly different CSF levels between HV and AD subjects; there also appeared to be a trend towards separation of the groups that increased with increasing length of the peptide. Aß1-42 appeared to be less correlated to other isoforms, with this correlation decreasing as the length of the Aß peptide decreased. Confirmation of these observations will require measurement of many more samples.


Key Point

  • Labelled internal standard peptides control for losses encountered during sample workup.
  • Values for amyloid-ß peptide levels determined by MS correlate very well with values obtained with ELISA assays
  • Multiple forms of the amyloid-ß peptide may be quantified in a single analysis.

 


   FOOTNOTES
 
Valentina Gelfanova is affiliated with Integrative Biology Division, Lilly Research Laboratories, Greenfield, IN.

Richard E. Higgs is affiliated with Discovery Statistics, Lilly Research Laboratories, Indianapolis, IN.

Robert A. Dean is affiliated with Diagnostic and Experimental Medicine, Lilly Research Laboratories, Indianapolis, IN.

David M. Holtzman is affiliated with Department of Neurology, Washington University School of Medicine, St. Louis, MO.

Martin R. Farlow is affiliated with Indiana University School of Medicine, Indianapolis, IN.

Eric R. Siemers is affiliated with Medical, Neurosciences Division, Lilly Research Laboratories, Indianapolis, IN.

Amechand Boodhoo is affiliated with Integrative Biology Division, Lilly Research Laboratories, Indianapolis, IN.

Yue-Wei Qian is affiliated with Integrative Biology Division, Lilly Research Laboratories, Indianapolis, IN.

Xiaohua He is affiliated with Integrative Biology Division, Lilly Research Laboratories, Indianapolis, IN.

Zhaoyan Jin is affiliated with Integrative Biology Division, Lilly Research Laboratories, Greenfield, IN.

Deborah L. Fisher is affiliated with Integrative Biology Division, Lilly Research Laboratories, Greenfield, IN.

Karen L. Cox is affiliated with Integrative Biology Division, Lilly Research Laboratories, Greenfield, IN.

John E. Hale is head of the Applied Biochemistry Group in the Integrative Biology Division at Lilly Research Laboratories, Greenfield, IN.


   References
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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