INTRODUCTION

Alzheimer's disease (AD)¹ is a progressive neurodegenerative disorder and the most common form of dementia (1). One of the neuropathological features of AD is the presence of amyloid deposits in senile plaques and in blood vessel walls (2, 3). These amyloid deposits are mainly composed of a 4-kDa protein, amyloid  protein (A), which contains 39-43 amino acid residues (4, 5, 6). A is derived from a 695-770-amino acid precursor, amyloid precursor protein (APP), through proteolytic processing (7, 8, 9). Since the initial isolation of A from amyloid deposits (4), various forms of A peptides have been reported. Both NH₂-terminally (5, 10, 11, 12, 13) and COOH-terminally (9, 14) truncated A peptides have been isolated and identified either from plaque cores (11), from neurofibrillary tangles (15), or from cerebrovascular amyloid fibrils (11, 16) from patients with AD or with Down's syndrome (5). The major A peptide in aqueous cerebral cortical extracts from AD brains has been reported as A-(1-40) (10). However, recent reports indicate that the insoluble amyloid in senile plaque cores is primarily A-(1-42) (11, 12) and that diffuse senile plaques are primarily A-(17-42) (17). These findings have been verified by measuring A in a 70% formic acid brain extract using sandwich enzyme-linked immunoabsorbent assay (18). In comparison, vascular amyloid is reported to be a mixture of A-(1-40) and A-(1-42) (11, 12).

The discovery that soluble A (sA) is a constituent of cerebrospinal fluid (CSF) (19, 20, 21) and cultured cell media (20, 22) indicates that A is a normal product of cellular metabolism of APP. The major form of A in biological fluids is A-(1-40). Both NH₂-terminal and COOH-terminal truncated sAs have been identified in pooled CSF specimens (21). A-(17-X) has also been detected in cultured cell media (22). The hypothesis that APP metabolism and A production play central roles in the pathogenesis of AD is supported by the observation that certain familial AD mutations cluster within or immediately adjacent to the A domain and these mutations influence APP processing and A production. For example, the APP_670/671 double mutations produce elevated A-(1-40) and A-(1-42) in vitro and in vivo (23, 24). Further, APP₇₁₇ mutations increase the relative amount of highly amyloidogenic A-(1-42/1-43) (27, 28), which may, in turn, recruit A-(1-40) to be deposited into amyloid plaques (29).

Numerous A species, in various concentrations, have been described in amyloid deposits from AD and control brain tissues (30). However, no significant difference that distinguishes AD patients and controls has been described in CSF studies of either sA peptide species (30) or concentration (20, 31, 32). We consider it likely that earlier methodologies used for A analysis may have failed to detect subtle variations in A levels between samples.

To investigate the production and metabolism of A in tissue, biological fluids, and cultured mammalian cell conditioned medium, we have developed a sensitive method for measuring sA and its variants using the strategy of microscale immunoaffinity capture (immunoprecipitation) and mass-specific identification (33, 34). The method relied on the combined approaches of immunoprecipitation and mass spectrometric analysis (IP/MS). The sA and its variants were selectively isolated by immunoprecipitation with anti-A mAbs. The identities of these isolated A peptides were determined by measuring their molecular masses using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (35). The relative signal intensities were used to estimate the concentrations of A. Using this approach, we have detected several novel A variants and have successfully quantified sAs in the conditioned media of cultured mammalian cells.

EXPERIMENTAL PROCEDURES

Mouse neuroblastoma cells (N2a), and cells stably transfected with Myc-epitope tagged wild type human APP₆₉₅ cDNA (N2a/APP₆₉₅) or APP₆₉₅ cDNA encoding Swedish mutant (N2a/APP_{695:595/596NL}) (36) were cultured in Dulbecco's modified Eagle's medium (high glucose)/Opti-MEM (1:1) containing 5% fetal bovine serum and 1% penicillin/streptomycin. All cell culture media were obtained from Life Technologies, Inc.. Geneticin (G418) was added to the growth media for the stably transfected cells at a concentration of 0.2 mg/ml. Cells were plated in 100-mm tissue culture dishes and grown in 5% CO₂ in a 37 °C incubator.

N2a cells were plated in multiwell cell culture plates (12-well plates, Becton Dickinson Labware, Franklin Lakes, NJ) and grown in 1.5 ml of growth medium until approximately 90% confluent. The growth medium was discarded, and cells were washed with phosphate-buffered saline (Life Technologies, Inc.). After washing, a serum-reduced medium (1 ml), consisting of Dulbecco's modified Eagle's medium (high glucose) and 0.2% fetal bovine serum, was added and cells were continually incubated in 5% CO₂ at 37 °C for 1 to 24 h. The conditioned media were harvested and centrifuged in a Beckman GS-6R centrifuge at 10,000 rpm (equal to 13,776 relative centrifugal force) for 5 min at 4 °C. The supernatants of centrifuged conditioned media were collected. Protease inhibitors (50 µg/ml antipain, 40 µg/ml bestatin, 2 mM EDTA-Na₂, 10 µM leupeptin, 1 µM pepstatin, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM L-1-chloro-3-[4-tosylamido]-7-amino-2-heptanone-HCl, 0.2 mM L-1-chloro-3-[4-tosylamido]-4-phenyl-2-butanone) and 0.01% NaN₃, were added immediately to prevent proteolytic degradation. Protease inhibitors were obtained from Sigma. The conditioned media supernatants were subjected to immunoprecipitation and mass spectrometric analysis.

Conditioned media supernatants (0.5-1.0 ml) were incubated with 1.0 µl of mAb 4G8 (2.5 mg/ml, anti-A-(17-24)) (37) or 6E10 (3.3 mg/ml, anti-A-(1-16)) (38) (Senetek, Maryland Heights, MO) in a rotator at 4 °C for 5-18 h. Protein G/A-agarose (3 µl, Oncogene Science, Inc., Cambridge, MA) was added, and rotational incubation was continued for an additional 3 h. The immunoprecipitated complex was collected by centrifugation in an Eppendorf Micro 5415C centrifuge at 10,000 rpm (equal to 8160 relative centrifugal force) for 2 min, and the supernatant was aspirated. The agarose beads were washed twice with ice-cold immunoprecipitation buffer (0.1% n-octylglucoside, 140 mM NaCl, 0.025% sodium azide, and 10 mM Tris-HCl, pH 8.0; Ref. 39) and once with 10 mM Tris-HCl, pH 8.0, containing 0.025% sodium azide. The samples were kept at 4 °C during the washing and centrifugation steps.

Mass Spectrometric Analysis of Immunoprecipitated A

Immunoprecipitated As were extracted with 3 µl of trifluoroacetic acid/water/acetonitrile (1:20:20, v/v/v) or formic acid/water/isopropanol (1:4:4, v/v/v), containing saturated -cyano-4-hydroxycinnamic acid (UV-laser desorption matrix) (40) and 200 nM bovine insulin (internal mass calibrant). 1.5 µl of the extraction solution was loaded onto the mass spectrometer sample probe and dried at ambient temperature. Mass spectra were measured using an UV-laser desorption/ionization time-of-flight mass spectrometer constructed at The Rockefeller University (41).

A-(12-28) (Sigma) was used as an internal standard. 1.0 µl of 20 µM A-(12-28) was added to 1.0 ml of conditioned media. The immunoprecipitation was carried out with 1.0 µl of mAb 4G8 and 3 µl of Protein G/A-agarose as described above. Formic acid/water/isopropanol (1:4:4, v/v/v), containing saturated -cyano-4-hydroxycinnamic acid were used to elute A peptides from immunoprecipitated complexes in all quantitative measurements. The quantitation curve was prepared by using synthetic peptides of A-(1-42), A-(1-40) (custom-made), and A-(1-28) (Sigma) with concentrations of 0.01-100 nM. Synthetic A peptides were dissolved in water/acetonitrile (1:1, v/v) at a concentration of 100 µM. The A peptide solution was diluted with medium of non-transfected N2a cells conditioned for 12 h in the presence of protease inhibitors. The diluted A peptide solutions (1 ml) were analyzed by IP/MS using mAb 4G8 (see above). The heights of peaks corresponding to A-(1-42), A-(1-40), A-(1-28), and A-(12-28) in mass spectra were measured and relative peak heights of A-(1-42), A-(1-40), and A-(1-28) to A-(12-28) were calculated. These relative peak heights were used to estimate the level of the corresponding sAs.

A Degradation in Cell Culture Media Conditioned by Non-transfected N2a Cells

Cell conditioned media of non-transfected N2a cells conditioned for 18 h were freshly prepared, as above but without addition of protease inhibitors. Synthetic human A-(1-40) or A-(1-42) (50 nM) was added to freshly harvested conditioned media and incubated at 37 °C for 22 h. At the end of the incubation period, 1 ml of medium was collected and protease inhibitor mixture (see above) was immediately added to the sample media. The sample was split, and 0.5 ml of aliquots were immunoprecipitated with either mAbs 4G8 or 6E10 (see above). A-related peptides were analyzed by MALDI-TOF-MS using trifluoroacetic acid/water/acetonitrile (1:20:20, v/v/v) as elution buffer (see above).

RESULTS

Endogenous Murine A and Its Variants Are Detected from Non-transfected Mouse Neuroblastoma Cells

Conditioned medium from non-transfected N2a cells was used as a control to examine the specificity of IP/MS for human A. 1 ml of conditioned medium was immunoprecipitated by mAbs 4G8 and 6E10 (separately) with Protein G/A-agarose beads and the immunoprecipitated peptides were analyzed by MS. Using mAb 4G8, we observed a series of peaks in the spectrum (Fig. 1, A and B). The molecular masses of the observed peaks indicated that they correspond to the amino acid sequences of murine A peptides (42). A summary of molecular masses of the observed peaks and the calculated molecular masses and amino acid sequences of their corresponding murine A peptides are provided in Table I. Although mAb 4G8 was raised against the amino acid sequence of human A, we have successfully detected murine A. The mAb 4G8 detects epitope between amino acids 17-24 of A, a sequence conserved between murine and human As (Fig. 2). In contrast, mAb 6E10 is human-specific and fails to recognize murine As (Fig. 1C). The amino acid sequences of murine A and human A differ at amino acids 5, 10, and 13 (Fig. 2). The differences in amino acid sequence also contribute to the differences in their molecular masses and made it possible to differentiate human and mouse A species by mass spectrometry.

MALDI-TOF-MS spectrum of endogenous murine soluble A and its variants detected from 1 ml of non-transfected N2a cell conditioned medium (24 h) by IP/MS using mAbs 4G8 (A and B), and 6E10 (C).

Table I. Soluble murine A and its variants detected from cell culture medium conditioned with non-transfected N2a cells by IP/MS using mAb 4G8 Mr (observed) A Mr (calculated) Amino acid sequence 1        10        20        30        40 4233.5a 1 -40 4233.8 DAEFGHDSGFEVRHQKLVFFAEDVGSNKGAIIGLMVGGVV 4035.4a 1 -38 4035.5 DAEFGHDSGFEVRHQKLVFFAEDVGSNKGAIIGLMVGG 3691.8 1 -34 3691.1 DAEFGHDSGFEVRHQKLVFFAEDVGSNKGAIIGL 3577.7 1 -33 3577.9 DAEFGHDSGFEVRHQKLVFFAEDVGSNKGAIIG 3459.4a 5 -36 3458.9     GHDSGFEVRHQKLVFFAEDVGSNKGAIIGLMV 3227.3 5 -34 3228.6     GHDSGFEVRHQKLVFFAEDVGSNKGAIIGL 3170.9a 11 -40 3170.7           EVRHQKLVFFAEDVGSNKGAIIGLMVGGVV 3070.0a 11 -39 3071.6           EVRHQKLVFFAEDVGSNKGAIIGLMVGGV 2973.6a 11 -38 2972.5           EVRHQKLVFFAEDVGSNKGAIIGLMVGG 2916.0a 11 -37 2915.4           EVRHQKLVFFAEDVGSNKGAIIGLMVG 2628.9 11 -34 2628.0           EVRHQKLVFFAEDVGSNKGAIIGL 2515.0 11 -33 2514.9           EVRHQKLVFFAEDVGSNKGAIIG 2102.8 11 -28 2103.4           EVRHQKLVFFAEDVGSNK 1975.2 11 -27 1975.2           EVRHQKLVFFAEDVGSN 3042.1a 12 -40 3041.6            VRHQKLVFFAEDVGSNKGAIIGLMVGGVV 2844.2a 12 -38 2843.3            VRHQKLVFFAEDVGSNKGAIIGLMVGG 2788.6a 12 -37 2786.3            VRHQKLVFFAEDVGSNKGAIIGLMVG 2499.2 12 -34 2498.9            VRHQKLVFFAEDVGSNKGAIIGL 2386.2 12 -33 2385.7            VRHQKLVFFAEDVGSNKGAIIG 2391.8a 17 -40 2392.8                 LVFFAEDVGSNKGAIIGLMVGGVV a  A 16-Da mass increase was observed when formic acid/water/isopropanol were used for elution.

Amino acid sequence alignment of human and murine A.

Human A and Its Variants Can Be Detected and Distinguished in Conditioned Medium of Cells Expressing Human APP

Cultured mammalian cells constitutively produce and secrete A. The profile of total soluble A expressed by cultured cells was characterized using mouse neuroblastoma (N2a) cells, which were stably transfected with human APP cDNAs encoding either wild type (N2a/APP₆₉₅) or Swedish familial AD mutant APP (N2a/APP_{695:595/596NL}). These mouse neuroblastoma cells were incubated in serum-reduced media (0.2% fetal bovine serum) for 24 h. The sA peptides secreted into the cultured media (0.5 ml) were analyzed by immunoprecipitation with anti-A mAbs and MALDI-TOF-MS. The mass spectrum resulting from IP/MS analysis of A peptides from N2a/APP_{695:595/596NL} cell conditioned medium using mAb 4G8 is shown in Fig. 3. The identities of peaks were assigned according to the measured molecular masses and several criteria (see below) and are identified in the figure according to human A amino acid sequence numbers. In addition to the species containing A amino acid sequences 1-40 (A-(1-40)) and 1-42 (A-(1-42)) (the major components found in senile plaque of AD), we observed several short forms of A-related peptides. A summary of these A-related peptides is given in Table II. A similar A profile was observed for cells expressing wild type human APP₆₉₅ (N2a/APP₆₉₅) (data not shown).

MALDI-TOF-MS spectrum of human soluble A and its variants detected from 0.5 ml of N2a/APP_{695:595/596NL} cell conditioned medium (24 h) by IP/MS using mAb 4G8 (see "Experimental Procedures").

Table II. Soluble A and its variants detected from cell culture medium conditioned with N2a/APP770:670/671NL cells by IP/MS Mr (observed) A Mr (calculated) Amino acid sequence 1        10        20        30        40 4513.8a,b 1 -42 4514.1 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA 4330.0a,b 1 -40 4329.9 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV 4231.6a,b 1 -39 4230.7 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGV 4131.9a,b 1 -38 4131.6 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGG 4074.7a,b 1 -37 4074.5 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG 3787.0a 1 -34 3787.2 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGL 3673.5a 1 -33 3674.0 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIG 3615.8a 1 -32 3616.9 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAII 3390.6a 1 -30 3390.6 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGA 3262.4a 1 -28 3262.5 DAEFRHDSGYEVHHQKLVFFAEDVGSNK 3134.3a 1 -27 3134.3 DAEFRHDSGYEVHHQKLVFFAEDVGSN 2461.8c 1 -20 2461.7 DAEFRHDSGYEVHHQKLVFF 2315.0c 1 -19 2314.5 DAEFRHDSGYEVHHQKLVF 2167.3c 1 -18 2167.3 DAEFRHDSGYEVHHQKLV 2068.2c 1 -17 2068.2 DAEFRHDSGYEVHHQKL 1955.0c 1 -16 1955.0 DAEFRHDSGYEVHHQK 1827.0c 1 -15 1826.9 DAEFRHDSGYEVHHQ 1699.0c 1 -14 1698.7 DAEFRHDSGYEVHH 1561.8c 1 -13 1561.6 DAEFRHDSGYEVH 2972.9b,d 14 -42 2970.5              HQKLVFFAEDVGSNKGAIIGLMVGGVVIA 2786.0b,d 14 -40 2786.3              HQKLVFFAEDVGSNKGAIIGLMVGGVV 2649.8b,d 15 -40 2649.1               QKLVFFAEDVGSNKGAIIGLMVGGVV 2393.2b,d 17 -40 2392.8                 LVFFAEDVGSNKGAIIGLMVGGVV 2588.5b,d 14 -38 2588.0              HQKLVFFAEDVGSNKGAIIGLMVGG 2452.2b,d 15 -38 2450.9               QKLVFFAEDVGSNKGAIIGLMVGG 3170.9d 6 -34 3168.5      HDSGYEVHHQKLVFFAEDVGSNKGAIIGL 2916.8d 8 -34 2916.3        SGYEVHHQKLVFFAEDVGSNKGAIIGL 2243.7d 14 -34 2243.6               HQKLVFFAEDVGSNKGAIIGL 2104.8d 15 -34 2106.4                QKLVFFAEDVGSNKGAIIGL 1978.2d 16 -34 1978.3                 KLVFFAEDVGSNKGAIIGL 1848.5d 17 -34 1850.1                  LVFFAEDVGSNKGAIIGL 2858.4d 5 -29 2857.1     RHDSGYEVHHQKLVFFAEDVGSNKG 2672.0d 5 -27 2671.9     RHDSGYEVHHQKLVFFAEDVGSN 2515.1d 6 -27 2515.7      HDSGYEVHHQKLVFFAEDVGSN 2346.8c 2 -20 2346.6  AEFRHDSGYEVHHQKLVFF 2199.5c 2 -19 2199.4  AEFRHDSGYEVHHQKLVF 2053.5c 2 -18 2052.2  AEFRHDSGYEVHHQKLV 1712.2c 2 -15 1711.8  AEFRHDSGYEVHHQ 1583.8c 2 -14 1583.6  AEFRHDSGYEVHH 1446.5c 2 -13 1446.5  AEFRHDSGYEVH 1512.4c 3 -14 1512.6   EFRHDSGYEVHH 1999.4c 4 -19e 1999.2    FRHDSGYEVHHQKLVF 1851.9c 4 -18e 1852.0    FRHDSGYEVHHQKLV 1383.4c 4 -14 1383.4    FRHDSGYEVHH a  Detected by both mAbs 4G8 and 6E10. b  16 Da mass increase was observed when formic acid/water/isopropanol were used for elution. c  Detected by mAb 6E10 only. d  Detected by mAb 4G8 only. e  The molecular mass of A-(5-20) is identical to A-(4-19) and A-(5-19) is identical to A-(4-18).

The molecular masses of individual peptides (observed as peaks in the mass spectrum) were measured and used to determine the identities of the these peptides. Several criteria were used for determining the identities of A peptide variants. (i) The peak observed in the spectrum of cell culture medium conditioned with N2a/APP_{695:595/596NL} cells (or N2a/APP₆₉₅ cells) should not exist in the control spectrum, which resulted from assay of medium conditioned by non-transfected N2a cells. (ii) The measured molecular mass of that peak should match the calculated molecular mass of A peptide(s) based on the human A amino acid sequence. (iii) The tentative matched A peptide should contain the epitope site for the mAb used in the immunoprecipitation. (iv) If the predicted A peptide contains a methionine residue, then its mass should increase by 16 Da when formic acid/water/isopropanol is used as elution buffer, since the methionine residue in A peptide at position 35 is readily oxidized under these conditions (42).

The results from IP/MS analysis with mAb 4G8 indicated that A peptides containing the 4G8 epitope can be identified. To obtain a more complete peptide profile for sA in cultured neuroblastoma cells expressing human APP, IP/MS analysis was also carried out using a human A specific antibody, mAb 6E10. Again, A-(1-42), A-(1-40), A-(1-34), A-(1-28), and other long forms of A-related peptides were detected, similar to the results obtained with mAb 4G8. A series of peaks with different masses from those in Fig. 3 were observed in the mass range of 1000 to 2500 Da (Fig. 4). These peaks corresponded to A related peptides with further truncated carboxyl termini (A-(1-13) to A-(1-20)), or with both carboxyl- and amino-terminal truncations (A-(2-13) to A-(2-20), A-(4-14), A-(4-18), and A-(4-19)) (Table II). In addition to identifying A-related peptides, our results indicated directly that the epitope site of mAb 6E10 is within amino acid sequence of 4-13 of A.² These A species were also observed from cell culture media conditioned with N2a/APP₆₉₅ cells (data not shown). Surprisingly, we identified a total of 44 different secreted human A-related peptides (Table II).

MALDI-TOF-MS spectrum of human soluble A and its variants detected from 0.5 ml of N2a/APP_{695:595/596NL} cell conditioned medium by IP/MS using mAb 6E10 (see "Experimental Procedures" and Fig. 1 legend).

Comparing the results from cell culture medium of N2a/APP_{695:595/596NL} cells (Figs. 3 and 4 and Table II) with results from cell culture medium of non-transfected N2a cells (Fig. 1 and Table I), peptides corresponding to human A sequence were uniquely observed only in N2a/APP_{695:595/596NL} cells (Figs. 3 and 4) and were absent in the control (Fig. 1). Finally, and in contrast to the results from mAb 4G8 IP/MS, no murine A or variants were detected in the spectrum resulted from the IP/MS analysis of cell culture medium conditioned with non-transfected N2a cells when mAb 6E10 was used (Fig. 3c). These results confirm the specificity of the mAb 6E10 for human A.

The Sensitivity and Dynamic Range of IP/MS for Measuring A Peptides

Four synthetic human A peptides, A-(1-42) (4514.1 Da), A-(1-40) (4329.9 Da), A-(1-28) (3262.5 Da), and A-(12-28) (1955.2 Da), were used in this study. A-(1-42) and A-(1-40) were chemically synthesized, and their concentrations were determined by amino acid analysis. A-(1-28) and A-(12-28) were obtained commercially (Sigma). A series of dilutions of each peptide was prepared ranging from 100 to 0.01 nM for peptides A-(1-42) (450 ng/ml to 45 pg/ml), A-(1-40) (430 ng/ml to 43 pg/ml), and A-(1-28) (320 ng/ml to 32 pg/ml) using serum-reduced media conditioned by non-transfected N2a cells for 12 h and immediately treated with protease inhibitors upon harvest. Conditioned medium of N2a cells was used to mimic the conditions used in a time-course study (see below). Only a small amount of endogenous murine A was produced within 12 h and did not interfere with the measurement of the human A peptides. A-(12-28), as internal standard, was added to each of the diluted solutions with a constant concentration of 20 nM prior to immunoprecipitation. We used A-(12-28) peptide as the internal standard because it appears not to be produced naturally (we did not detect it in our analysis of secreted peptides from N2a cells). The effects of experimental variations (e.g. signal variations due to the absolute amount of Protein G/A-agarose beads or to the laser intensity used during the MS measurement) on the resulting MS peak height was corrected by normalization to the internal standard, i.e. the peak height of A-(12-28). The peak heights of A peptides were measured, and the relative peak heights of A-(1-42), A-(1-40), and A-(1-28) to A-(12-28) were calculated. These relative peak heights were used to evaluate the reproducibility and sensitivity of IP/MS in measuring A peptides.

Fig. 5 depicts a series of mass spectra resulting from immunoprecipitation of five different concentrations of A peptides using mAb 4G8. The peaks corresponding to A-(1-42), A-(1-40), A-(1-28), and A-(12-28) are labeled and also indicated by dotted lines. Other peaks appear in the spectra, especially in spectra of low A concentrations. These peaks correspond to endogenous murine A peptides. The spectra in Fig. 5 clearly show that As can be detected at levels as low as 0.1 nM (0.01 nM for A1-28), with a signal-to-noise level greater than 2. This corresponds to about 0.4 ng/ml for A-(1-40). The average relative peak height for each A peptide obtained by three independent measurements are plotted versus concentration in Fig. 6. This plot indicates that IP/MS has a very wide dynamic range (linearity between the relative peak height and the A concentration) for A measurement. The correlation coefficients between the relative peak heights and peptide concentrations are 0.9993 for A-(1-28) in the concentration range of 0.01 nM to 100 nM, 0.9992 and 0.9997 for A-(1-40) and A-(1-42), respectively, over the concentration range of 0.1 nM to 100 nM. The standard deviations (S.D.) based on three measurements are plotted in Fig. 6 with error bars, which indicate standard errors of the means. The coefficient of variation (the percentage of standard deviation to the measured mean) is 21.8%, averaging all data in this study.

MALDI-TOF-MS spectra of synthetic human A-(1-42), A-(1-40), and A-(1-28), at concentrations of 100, 10, 1, 0.1, and 0.01 nM in cell culture media.

Relative peak height as a function of A concentration.

A Time-course Measurement of A Protein and Its Variants Secreted by Cultured Cells

The accumulation of A-(1-40), A-(1-42), and several A fragments produced by cultured cells were carefully monitored by IP/MS as a function of time. N2a/APP_{695:595/596NL} cells were propagated in 1.5 ml of growth medium in 12-well tissue culture plates. To prepare conditioned media, cell monolayers were washed three times with phosphate-buffered saline and then placed in 1 ml of serum-reduced medium and incubated at 37 °C, 5% CO₂ for times between 1 h and 30 h. The harvested media were treated immediately with a protease inhibitor mixture (see above) to prevent proteolytic degradation. As were immunoprecipitated together with an internal standard, A-(12-28), by mAb 4G8 and analyzed by MS. The mass spectra from four time points are shown in Fig. 7. In order to reduce the fluctuation in the A measurements that is caused by the variation in cell number in each measurement, two separate measurements were performed for each time point. The average peak heights of A peptides relative to standard (A-(12-28)) and measured A peptide concentrations using standard curve (Fig. 6) were plotted versus time to view the relationship between the truncated A and A-(1-40/1-42) (Fig. 8). We observed that the total level of As increased during the 20-h incubation time. The data shown in Fig. 8 represent the integrated values of sA peptides in cell culture media. In other words, these results represent net A levels, which are presumably determined by the balance of production and degradation of A in the cell culture media. The rate of A turnover cannot be determined from our current data due to the continuous production of A by cells.

Mass spectra of A and variants obtained during a 30-h time course of N2a/APP_{695:595/596NL} cell conditioned media.

Proteases in Conditioned Medium Degrade Exogenous A to an Insignificant Degree

To examine the origin of the short A peptides observed in cell culture media, we studied A proteolysis in conditioned medium of non-transfected N2a cells. Conditioned medium was freshly prepared by conditioning serum-reduced medium with non-transfected N2a cells. Synthetic human A-(1-40) (50 nM) was incubated at 37 °C for 22 h in the medium of cell conditioned for 18 h. The degraded A peptides were analyzed by IP/MS with mAbs 4G8 and 6E10. The representative mass spectra resulting from 0-h and 22-h samples of A-(1-40) are shown in Fig. 9. We observed very low levels of truncated A peptides after 22 h of incubation (Fig. 9, B and D). These degraded A peptides, primarily generated by proteolytic cleavages after lysine 28, histidine 13/14, and hydrophobic amino acids (phenylalanine 19/20, glycine 33, and leucine 34), are present at low levels in comparison to full-length A-(1-40).

A peptide fragments generated by proteolytic degradation of synthetic human A-(1-40) peptide in non-transfected N2a cell conditioned medium were identified by IP/MS analysis.

DISCUSSION

Amyloid  Protein and Its Variants Are Readily Detected by Immunoprecipitation/Mass Spectrometric Analysis

The present study reports a comprehensive profile of sA secreted by cultured cell lines using a novel IP/MS method. Sixty-four A-related peptides (44 from human and 20 from murine A sequences) have been identified from immunoprecipitated conditioned cell culture media without further purification. Compared to other methodologies used to measure sA, the IP/MS has several significant advantages. High affinity antibodies were utilized to specifically purify and enrich the peptides of interest. This enrichment enabled us to analyze the peptides of interest directly from a small volume of biological fluid with low A concentrations. This technique should be very useful in the analysis of sA from human specimens (e.g. CSF). Accurate molecular masses of A peptides were determined in the present measurement. Because highly specific A monoclonal antibodies were utilized, these accurate molecular masses provide essential information regarding the identities, amino acid composition, and sequence length of A and its related variant peptides. Another important feature of this mass-specific detection assay is the ability to simultaneously analyze complex mixtures of As in biological samples in a single assay. Hence, both the molecular masses and the concentrations of sAs can be determined in a single measurement using appropriate A-peptide analogs as internal standards with appropriate standard curves (see below).

Characterization of Amyloid  Protein and Its Variants

A-(1-40) has been detected in all cell lines studied, including cells transfected with human APP and non-transfected mouse cells. A-(1-40) is the predominant form isolated from cerebral cortical deposits (9), vascular deposits (10, 15), cerebrospinal fluid (18), human mixed-brain cell culture (18), and other cultured cell lines. A-(1-42), on the other hand, has only been detected in the medium of cells transfected with human APP cDNAs. In addition, more than 40 truncated A peptides have been identified from cell culture media conditioned with N2a/APP_{695:595/596NL} or N2a/APP₆₉₅ cells (Table II). These A peptide fragments can be classified into five different groups according to the patterns of the peptides: (i) peptides with truncated carboxyl termini (these range from A-(1-13) to A-(1-39)); (ii) peptides with truncated amino termini (A-(14-42), A-(14-40), A-(15-40), and A-(17-40) have been observed); (iii) peptides with ragged NH₂ termini and new COOH termini, possibly resulting from internal cleavage of A (these include A-related peptides ending with Gly³⁸, Leu³⁴, and Asn²⁷ at their COOH termini); (iv) peptides with ragged COOH termini and a new NH₂ termini, possibly resulting from internal cleavage of A (these include A-related peptides beginning with Ala², Phe⁴, Arg⁵, and His⁶ at their NH₂ termini); (v) other internal peptide fragments of A (for example, peptide A-(3-14)). Several peaks that correspond to A-(1-38), A-(1-34), A-(1-28), A-(1-20), A-(1-19), and A-(1-14) were observed as prominent peaks in the mass spectra (see Figs. 3 and 4).

In contrast to the A-related peptides observed from cell culture media conditioned with N2a/APP_{695:595/596NL} and N2a/APP₆₉₅ cells, a different proteolytic digestion pattern was observed from cell culture media conditioned by non-transfected N2a cells. The predominant murine A peptide fragments observed in this study apparently resulted from cleavages around amino acid residue Glu¹¹. These peptides start at either Glu¹¹ or Val¹² at their NH₂ termini. The different termini in A species of human versus mouse As may be due to alternative proteolytic events that are directed by the differences in human and mouse A peptide sequences (Fig. 2).

We have observed a complex set of A peptides from cultured cell media. We are quite confident that these peptides are not artificially produced by proteolysis of full-length 1-40/42 peptides in the medium after harvest, or during our antibody incubation steps. In support of this view, we have demonstrated that synthetic A-(1-40) and A-(1-42) peptides are not degraded upon addition to the medium of non-transfected N2a cells conditioned for 12 h and subsequently subject to IP/MS (Fig. 5), in the existing of protease inhibitors. Furthermore, these A peptide fragments are also not generated extrocellularly by proteolytic degradation as consequence of proteolytic activity in fetal bovine serum that was used in the conditioning medium (data not shown). The mechanisms by which these A-related peptides are generated is presently not clear. Nevertheless, clarification of the relationship of these short A forms and longer A-(1-40/1-42) forms may provide some insight into the production, metabolism and deposition of A in AD.

Broad Concentration Ranges of A Peptide Can Be Measured by IP/MS

In conventional immunoprecipitation, quantitative recovery of a specific protein (antigen) is the primary goal. This is achieved by using excess antibody and excess precipitation reagents. Normally, 10 µl of mAb and 15 µl of Protein G/A-agarose beads are used for immunoprecipitation from 1 ml of cellular extraction or cell culture media (according to the manufacturer's protocol, Oncogene Science, Inc). In comparison, our present approach uses only 1 µl of mAb and 3 µl of Protein G/A-agarose beads for immunoprecipitation. The small amount of immunoprecipitation reagent used here is intended to reduce the final elution volume and allow direct mass spectrometric measurement of antigens. We have attempted to achieve quantitative sampling of the antigens rather than 100% immunoprecipitation of them. For quantitative sampling (using small amount of precipitation reagent), a proper ratio of mAb and precipitation reagent is essential to provide high binding capacity and high detection sensitivity for the peptide (antigen) of interest. If the ratio is too low, the peptide binding capacity will be reduced and only the most abundant species and some minor species (possibly certain polymeric A peptides) with substantially higher avidity to the mAbs will be detected. If the ratio is too high, the sensitivity will be reduced by the increased occupation of free antibodies on the binding site of the precipitating reagent. Therefore, the amount of mAb and Protein G/A-agarose bead and the ratio of these two reagents was carefully optimized. Using a mAb/precipitation reagent ratio of 1:3 (v/v), we have been able to measure a very broad concentration range of A peptides (from 0.01 nM to 100 nM) (Fig. 6). The concentration of A in CSF, plasma, and cultured cells has been reported to be in the range of 0.6-8 nM, 0.1-0.3 nM, and 0.7-1 nM, respectively (as the black bar indicates in Fig. 6) (18, 31). The results from our experiments suggest that IP/MS will be useful in measuring A and its variants in human body fluids. The sensitivity of IP/MS is comparable with enzyme-linked immunosorbent assay, which is 0.1 ng/ml (0.02 nM) for As (18).

Our data also indicate that different A peptides have very different signal intensities when assayed by mass spectrometry. For example, at the same concentration, A-(1-28) results in a peak intensity that is almost 5-fold higher than that of A-(1-40). These different peak intensities are likely due to the different ionization efficiencies of the different peptides in the mass spectrometric measurement. These different responses may also arise from differences in solubility of each individual peptide and/or differences in affinity for the antibodies. However, these experimental effects on the quantitative measurement of A can be corrected by using appropriate A-peptide analogs as internal standards and standard curves for each peptide as present in the mixture. Our data strongly suggest that it is inappropriate to compare the concentrations of any two peptides from their peak heights in the mass spectrum. The concentration of a given peptide can only be measured using the relative peak height of this peptide to an internal standard and compared with a standard curve of the corresponding peptide. In the absence of a standard curve, the relative peak height can only be used to semi-quantitatively compare the levels of the same peptide.

The Concentrations of A-(1-40) Peptide and A-Related Peptides Increase in Parallel over 20 h in Cell Culture Media

The results of our time-course experiments indicate that the concentrations of both A-(1-40) and A-(1-42) increase over a 20-h period (Fig. 8). More interestingly, the major fragment peptides of A (for example, A-(1-34) and A-(1-28)) observed at late time points were also detected at very early time points and their amounts increased in proportion to the increasing amounts of A-(1-40). This result suggests two possibilities regarding the metabolism of these A fragment peptides. The first possibility is that these A fragment peptides are generated directly from APP by bona fide proteolytic processing events and are secreted simultaneously with A-(1-40) into the cell culture medium. The second possibility is that A is constantly degraded by proteases immediately after its secretion into the cell culture medium.

A Degradation in Conditioned Medium

Our analysis of degradation products of synthetic human A peptides revealed four primary cleavage sites (His¹³/His¹⁴, Phe¹⁹/Phe²⁰, Lys²⁸, and Gly³³/Leu³⁴) with three different endoprotease substrate specificities. These A peptides contribute to the low levels of certain A peptide variants normally observed in cell culture media of transfected cells. However, only a small portion of synthetic A-(1-40) peptide was degraded in conditioned medium over 22 h. A degradation by proteases released from cultured Chinese hamster ovary (CHO) cells and monkey kidney COS cells have been reported recently (44, 45). In the published studies (44), pulse-chase analysis revealed that the concentration of A in the CHO conditioned medium reaches a peak level about 6 h into the time course, and diminishes over the next 18 h. The inhibition of A degradation in CHO conditioned medium required the presence of all four major classes of protease inhibitors. Rapid degradation of A in COS conditioned medium has also been reported (26). A serine protease-₂-macroglobulin complex was described in the latter report as being responsible for A degradation in COS conditioned medium. This novel serine protease cleaves A primarily after hydrophobic amino acid residues (A residues 17-19 and 32-34) and degrades synthetic A-(1-40) almost completely in 16 h. This protease activity can be inhibited by certain types of serine protease inhibitors (26). In the present report, we demonstrated A degradation in N2a conditioned medium. Howeve