brain deposition of amyloid ( a ) peptides into neuritic plaques is one of the classical neuropathological hallmarks of alzheimer 's disease ( ad).1 a peptides are generated under physiological conditions by consecutive proteolytic cleavages of the amyloid precursor protein ( app ) by socalled  and secretases.2 a wellknown  nonamyloidogenic  app processing pathway involves cleavage within the asequence by secretase.3 , 4 recently , further cellular app processing pathways involving  and secretases , both cleaving full length app nterminally to the secretase cleavage site , were reported.5 , 6 
  a considerable number of a variants differing in the exact length of their amino and carboxy termini have been identified in amyloid plaques,7 in blood plasma,8 and in the cerebrospinal fluid ( csf).9 in particular , a peptides ending at ala(42 ) ( a42)10 , 11 and nterminally truncated a peptides starting with glu(3 ) , posttranslationally cyclized into pyroglutamic acid ( n3pe),12 , 13 or phe(4 ) are highly abundant in the parenchymal amyloid plaques in ad brain.1 well documented and widely accepted biomarkers of cerebral a accumulation , which have been incorporated into updated recommendations on diagnostic guidelines for alzheimer 's disease , are increased tracer signals on amyloid positron emission tomography ( pet ) imaging and low levels of soluble a42 in the csf.14 recently , the concentration ratio of the a peptides app669711/a142 in blood plasma , as measured by immunoprecipitation followed by mass spectrometry , was reported to be strongly and positively correlated with amyloid pet.15 a validated and reliable biochemical adsurrogate biomarker in blood would be highly advantageous for clinical routine , in particular since blood is much easier accessible than the csf . the peptide app669711 ( app770 numbering ) refers to an a40 peptide starting 3 residues upstream of the  classical  a sequence , that begins with asp(1 ) . the source of a340 in blood is currently unknown , neither is its role under physiological or pathological conditions . in cell culture supernatants of the chinese hamster ovary cell line 7pa2 , transfected with mutant app751 ( val  phe 717 ) , a340 was reported to be increased on inhibition of the site appcleaving enzyme 1 ( bace1 ) , suggesting that it results from an appprocessing pathway independent of bace1.16 herein , we describe the solidphase synthesis of the three nterminally elongated a peptides a338 ( 1 ) , a340 ( 2 ) , and a342 ( 3 ) ( table  1 ) . we also present a comprehensive biophysical characterization of the nterminally elongated peptides addressing peptide conformations and their aggregation into sheetenriched amyloid fibrils in vitro . moreover , we have identified a340 ( 2 ) to represent one of the baceinhibitorresistant a peptides produced from shsy5y cells transfected with wildtype human app . amino acid sequence and yields of the nterminally elongated a peptides a338 , a340 , and a342 ( 13 ) . [ a ]  yields after purification of the crude products by preparative hplc . several a variants have already been synthesized by various techniques including solidphase peptide synthesis ( spps).17 , 18 the synthesis of a peptides , elongated by three residues at the nterminus , was achieved by automated solidphase peptide synthesis on an abi  433a peptide synthesizer . attempts to obtain the a peptides 13 using a wang resin on polystyrene as a solid support , the standard fastmoc protocol from abi , and the oacylisopeptide method failed.17c17e however , when using a wang resin on polyethylene glycol as a solid support ( 0.1  mmol scale)17b in combination with a 10fold excess of fmocprotected amino acids ( 1  mmol ) , extended coupling times ( 35  min or 50  min),17b , 20 and a twofold coupling for the amino acids ser(26 ) and ala(30 ) , the a peptides 13 were obtained as major products ( see experimental section ) . after purification by preparative hplc , the a peptides 13 were isolated in good yields ( table  1 ) and high purities ( table  2 ) . the characterization of 13 was achieved by highresolution esims ( table  2 , supporting information ) . 
  analytical data for the a peptides 13 . [ a ]  retention time on analytical hplc ; column : vydac 208tp104 ( reversed phase c8 , 4.6250  mm ) ; flow rate : 1.0  ml  min ; eluent a : h2o with 0.1  % tfa ; eluent b : mecn with 0.1  % tfa . [ c ]  calculated monoisotopic mass . [ d ]  monoisotopic mass reconstructed by deconvolution of the esi mass spectra . [ e ]  gradient from 20  % to 50  % of eluent b in 30  min . [ f ]  gradient from 60  % to 90  % of eluent b in 30  min . for a338 ( 1 ) and a340 ( 2 ) the isolation after spps and purification by preparative hplc was achieved without any difficulty in a standard manner . however , the behavior of a342 ( 3 ) was very different . some of the a342 ( 3 ) precipitated already during cleavage of the crude peptide from the peg resin , indicating that more of the cleavage reagent ( tfa/1,2ethanedithiol / h2o / ipr3sih , 95:2:2:1 ) was required in order to remove the peptide completely from the resin . moreover , for hplc the standard polar gradient proved to be unsuitable . with water as major component of the mobile phase , a342 ( 3 ) formed undefined oligomers that were eluting from the column over a long period of time . using acetonitrile as major component in the hplc solvent mixture for the purification process , a342 ( 3 ) eluted from the column as a single peak after about 3  min ( table  2 ) . this procedure provided the nterminally elongated a peptides 13 in quantities of 1530  mg ( see experimental section ) . since the a peptides can misfold and give rise to aggregates leading to oligomers and fibrils , they are considered as peptides which are difficult to synthesize.19 therefore , the present synthesis of the a peptides 13 is remarkable with respect to scale and purity . several nterminally elongated peptides including a338 ( app669709 ) , a339 ( app669710 ) and a340 ( app669711 ) have been identified in human edtablood plasma.15 furthermore , the ratio of a340 to a142 ( app669711/a142 ) was proposed as a novel peripheral surrogate biomarker of amyloid deposition in the brain of ad patients.15b so far , nothing is known about the structure of the a340 peptide ( 2 ) and its potential to form insoluble aggregates . having the nterminally elongated a peptides 13 available by synthesis , we first sought to obtain insight into the overall structure of a340 ( 2 ) in solution by pulsefield gradient ( pfg)nmr experiments , which quantify the diffusion of molecules and thus allow quantification of their hydrodynamic radius ( table  3 ) . application of pfgnmr measurements to 70  m of freshly dissolved a340 ( 2 ) resulted in a translational diffusion coefficient of 5.910  cm  s.21 on the basis of this diffusion coefficient and the stokes einstein equation , a hydrodynamic radius r 
 h of 1.91  nm was calculated for a340 ( 2 ) . this hydrodynamic radius is consistent with an elongated , disordered peptide of 43 amino acids.22 next , we investigated the two nterminally elongated peptides , which end either at residue 38 ( a338 ) ( 1 ) or 42 ( a342 ) ( 3 ) . determination of the translational diffusion coefficients followed by calculation of r 
 h , resulted in values of 1.79 and 1.60  nm for a338 ( 1 ) and a342 ( 3 ) , respectively . despite the longer sequence , the r 
 h of a342 ( 3 ) was slightly smaller than that of a338 ( 1 ) and a340 ( 2 ) . this result suggests that a342 ( 3 ) is more compact than the peptides 1 and 2 . translational diffusion coefficients and hydrodynamic radii of a peptide variants obtained through pfgnmr diffusion measurements . [ c ]  translational diffusion coefficients were obtained after gradient calibration as described previously.21 [ d ]  the errors represent 95  % confidence intervals . it is known that a140 and a142 monomers in aqueous solution are mainly unstructured,23 , 24 but exhibit a tendency to adopt hairpin conformations.24 , 25 to gain insight into the monomeric structure of the nterminally elongated a peptides at single residue level , 2d homonuclear proton nmr spectra were measured for freshly prepared a338 ( 1 ) , a340 ( 2 ) , and a342 ( 3 ) samples ( ca . 70  m , ph  7.4 buffered with 20  mm sodium phosphate ) . the sequencespecific protonresonance assignments of all three peptides were obtained through the analysis of twodimensional tocsy and noesy spectra . we then compared the tocsy spectra of the three peptides 13 ( figure  1 ) . these peptides , which all start at position 3 and only differ at the cterminus , show a nearly complete overlay of their tocsy spectra , with differences being restricted to the immediate cterminal regions ( figure  1  b ) . in contrast , the chemical shifts of a340 ( 2 ) and a140 revealed some interesting differences beyond the presence of the four additional peaks , which correspond to lys(2 ) , met(1 ) , asp(1 ) , and ala(2 ) ( figure  1  a ) . in particular , the cross peaks of residues glu(3 ) , phe(4 ) , arg(5 ) , and to a lesser extent asp(7 ) , gly(9 ) , and tyr(10 ) showed perturbations in their hn chemical shifts . moreover , the chemicalshift deviations were not limited to the nterminal region of a , but also included residue ser(26 ) , which points to a longrange effect of the nterminal elongation . taken together , the nmr data demonstrate that nterminally elongated a peptides are disordered in solution and that nterminal elongation influences the backbone conformation of a particularly in , but not limited to , its nterminal region . effects of nterminal elongation on the structure of a peptides as revealed by 2d protonproton nmr spectroscopy . a ) superimposed tocsy spectra of a140 ( purple ) and a340 ( 2 ) ( red ) along with crosspeak assignments . some additional peaks are observed in the spectrum of a340 ( 2 ) ( m1 , k2 , d1 , a2 ) . peaks with strong displacement are highlighted by arrows ( e3 , f4 , r5 ) . b ) superposition of the tocsy spectra of a338 ( 1 ) ( cyan ) , a340 ( 2 ) ( red ) , and a342 ( 3 ) ( green ) show a nearly perfect fit except in their differing cterminal region . c ) combined hn and ha chemicalshift difference between a140 and a340 ( 2 ) indicate local conformational changes in the nterminal region as well as a longrange effect on residue ser(26 ) ( highlighted by asterisk ) . the nterminal region of a peptides is highly important for its aggregation and toxicity.26 , 27 , 28 we therefore decided to investigate the impact of nterminal elongation on the aggregation behavior of a peptides using a set of biophysical techniques . first , we performed an nmrbased monomerconsumption assay to quantify the timedependent conversion of a monomers to nmrinvisible aggregates . the 1d proton nmr spectra of a samples were measured before and after 24  h of incubation in aggregationprone conditions ( 37  c , gentle stirring ) . the concentration of monomeric a was estimated from the nmr signal intensity using dss ( 4,4dimethyl4silapentane1sulfonic acid ) as internal reference . immediately after dissolving and before incubation , the monomeric concentrations of a338 ( 1 ) and a340 ( 2 ) , as estimated by nmr spectroscopy , were very close to their nominal concentration , indicating that they were almost fully solubilized ( figure  2 ) . on the other hand , the initial monomeric concentration of a342 ( 3 ) was approximately only 60  % of its nominal concentration , reflecting its limited solubility . after 24  h of incubation , the monomer concentrations of a338 ( 1 ) , a340 ( 2 ) , a342 ( 3 ) , and a142 decreased to 11 , 6 , 15 , and 6  % of the initial nominal concentrations , respectively ( figure  2 ) . . further comparison of the data for a342 ( 3 ) and a142 showed that nterminal elongation resulted in a decreased a peptide monomer loss . after 1  day of aggregation , the monomer concentration , determined by nmr spectroscopy , significantly decreased in all a peptide variants of this study . the relative errors in peptide concentration estimated from duplicate nmr measurements are smaller than 1  % . next , we monitored if the secondary structures of the nterminally elongated a peptides ( 13 ) were altered during aggregation . to this end , we used uv circular dichroism ( figure  3  a ) . before aggregation , a338 ( 1 ) , a340 ( 2 ) , a342 ( 3 ) , and a142 showed cd spectra with negative minima around 200  nm , which are characteristic for disordered polypeptide conformations . in agreement with their incomplete solubilization , evidenced by nmr spectroscopy ( see above ) , the cd spectra of a342 ( 3 ) and a142 had less negative ellipticities at 200  nm . in addition , larger negative ellipticities were observed around 220  nm , indicating the copresence of sheetrich aggregates in these samples . after 24  h of aggregation , all four a peptide variants exhibited structural conversion from randomcoil to sheet ( figure  3  b ) . the spectra of the aggregated a142 , and to a lesser degree a342 ( 3 ) , contained strong negative peaks around 217219  nm , indicative of extended sheet segments . in contrast , the cd spectra of aggregated a338 ( 1 ) , and in particular a340 ( 2 ) , showed significant red shifts of this negative peak to about 222224  nm , suggesting that the aggregated species of these two a variants may contain some typeii turn structures . a ) the freshly prepared a peptide solutions showed predominantly coillike structures with a negative band around 200  nm . b ) after aggregation , a prominent coiltobeta transition occurred in all studied a peptide variants . the cd intensity difference in b ) cd spectroscopy showed that the nterminally elongated a peptides ( 13 ) aggregate into sheetrich structures . to test if the formed aggregates are amyloid fibrils , we used the amyloidspecific dye thioflavin  t ( tht ) . the fluorescence intensity of tht is low when in solution or bound to less stable aggregation intermediates , but is very high when bound to amyloid fibrils.29 all three nterminally elongated a peptide variants exhibited a high rate and amount of aggregation ( figure  4 ) . after only one hour of incubation in aggregationprone conditions a significant rise in tht fluorescence emission intensity was observed for a338 ( 1 ) , a340 ( 2 ) , and a342 ( 3 ) . in case of a142 , sizeable tht fluorescence intensity was already present in freshly prepared samples , indicating the incomplete solubilization of preformed aggregates . to further investigate the ability of nterminally elongated a peptides to form amyloid fibrils , we examined the aggregated samples by electron microscopy . the peptide a338 ( 1 ) formed fibrillar aggregates of various morphologies ( figure  5 ) . the majority of the aggregates were twisted pairs of long fibrils , while also some straight fibrils with or without lateral association were observed . in the a340 ( 2 ) sample , fewer but longer fibrils were observed and they were predominantly twisted . in contrast , both a342 ( 3 ) and a142 formed a mixture of short curved protofibrils and long twisted fibrils ( figure  5 ) . fibrillar sheetrich aggregation of a peptides as followed by a thioflavin  t ( tht ) fluorescence assay . all three nterminally elongated variants of a peptides show a high rate and degree of thtreactive aggregation . electron microscopic images of the a peptides 13 and a142 after 24  h incubation under aggregationprone conditions ( 37  c , gentle stirring ) reveals their ability to form amyloid fibrils . in order to evaluate the effect of nterminal elongation on the kinetics of a peptide fibrillar aggregation , we monitored the temporal development of tht fluorescence in ax40 and ax42 peptide variants in a realtime manner . at a low peptide concentration of roughly 10  m , incubation in mild aggregation conditions ( room temperature , with agitation ) resulted in a gradual increase in the tht fluorescence intensities of table  4 summarizes the kinetic parameters obtained from the analysis of the tht traces according to a logistic model.26 , 30 the rate constants suggest that the nterminal extension by three residues promoted the fibrillar aggregation of a40 and a42 . it should , however , be noted that the fluorescence signal intensities of a340 ( 2 ) frequently rose to the maximum detection limit of the fluorimeter , hence precluding a reliable quantitative analysis of its aggregation curves . furthermore , the comparison of the aggregation kinetics between a142 and a342 ( 3 ) was complicated by the presence of preformed aggregates in a142 samples as indicated by the relatively high tht intensities at initial time points . realtime monitoring of thioflavin  t ( tht ) fluorescence during a peptide incubation under aggregationprone conditions ( rt , 10  m of a peptide , gentle stirring ) . the nterminally elongated variants of a peptide ( 2 and 3 ) showed accelerated aggregation when compared to their corresponding a1x peptide analogues . averages of three separate tht traces are shown , except of a340 ( 2 ) for which the tht intensity in all but one trace quickly rose to the maximum detection limit of the fluorimeter . kinetic parameters of a peptide fibrillar aggregation obtained from analysis of the timedependent increase in tht fluorescence intensities . [ a ]  f 
 0 and f 
 lim denote the initial and final fluorescence intensities ; t 
 1/2 represents the time for a peptide samples to reach half of their final tht fluorescence intensities ; k denotes a secondorder rate constant governing the growth of thtreactive aggregates according to the equation : df / dt = k 
 f  ( f 
 limf ) . [ c ]  for a142 , t 
 1/2 was not determined since the initial fluorescence level ( f 
 0 ) was already higher than half of the final level ( f 
 lim ) . for further insight into the kinetics of a peptide aggregation , we measured the rate of monomer loss during a peptide aggregation within the nmr tube ( 37  c , without agitation ) . the aggregation reaction was initiated by addition of aggregation seeds , which were prepared from a mixture of amyloid aggregates from the a peptide variants under investigation . the highest rates of monomer loss were observed for a342 ( 3 ) and a142 , followed by a338 ( 1 ) and then by a340 ( 2 ) and a140 ( figure  7 ) . the comparison of the monomerconsumption profiles , especially in the initial parts , suggests that the nterminal extension of a peptides by three residues did not have any significant impact on the monomerincorporation rates into previously formed aggregates . more rigorous kinetic experiments are , however , required before this conclusion can be confirmed . nmrbased monitoring of a peptidemonomer consumption during aggregation induced by addition of aggregation seeds ( 37  c , without agitation ) . no significant differences were observed between a142 and a342 ( 3 ) or a140 and a340 ( 2 ) , especially in the initial parts of the monomerconsumption profiles . taken together , our data indicate that nterminally elongated a peptides ( 13 ) are disordered in solution and rapidly form sheetrich fibrillar aggregates . from the comparison of a3x with a1x we suggest that nterminal elongation increases the propensity for aggregation , although the monomer incorporation rate to preformed aggregates does not seem to be significantly affected . this is in apparent contrast with a very recent study , in which the impact of nterminal elongation , ranging from 540 amino acids in length , on the aggregation kinetics of recombinant variants of human a42 was investigated.31 all of the nterminally extended forms of a42 under investigation were shown to be able to form amyloid fibrils similar to those formed from a142 , but with lower rates . the apparent discrepancy between these two studies may result from differences in experimental procedures such as solubilization of a peptide prior to aggregation assays or the different lengths of the nterminal extensions . six years ago , we reported that treatment of shsy5y cells overexpressing wild type human app with membraneanchored tripartite bace1 inhibitors reduced the overall cellular generation of a peptide , but at the same time led to a relative increase in specific a peptide variants displaying isoelectric points ( pi ) different from that of known a peptides starting with asp(1 ) , that is , pi of approximately 5.4 according to 2dwestern blot analysis.32 mass spectrometry data indicated that the bace1inhibitorresistant a peptides comprise primarily ntruncated peptides starting with arg(5 ) , and have a pi of approximately 6.5 according to a novel capillary isoelectricfocusing ( cief ) immunoassay.16 , 18 , 33 , 34 , 35 in addition , a minor fraction of bace1inhibitorresistant a peptides with a pi of approximately 6.0 was observed in supernatants of app overexpressing shsy5y cells ( see figure  2 in ref . although a240/a340 were the most obvious candidates on the basis of their predicted isoelectric points , no evidence for the presence of such nterminally truncated peptides was found using immunoprecipitation followed by mass spectrometry.18 however , when considering the occurrence of nterminally elongated a variants we could now assign prominent signals to a338 ( 1 ) and a340 ( 2 ) , and identify the latter as the main constituent of the minor fraction of bace1inhibitorresistant a peptides in the shsy5y cell supernatant ( figure  8) . given that a240/a340 and a340 ( 2 ) have a virtually identical predicted isoelectric point , the question arose whether these two a classes can be separated by isoelectricfocusing techniques . the availability of synthetic a340 ( 2 ) enabled us to directly compare the isoelectric points of a140 , a240 , and a340 ( 2 ) by cief immunoassay ( figure  9 ) . most importantly , nterminally elongated a340 ( 2 ) can be resolved from nterminally truncated a240 by isoelectric focusing due to subtle differences in net charge , providing a powerful tool to distinguish these two a classes of biological and potentially pathological relevance . massspectrometric identification of the a peptides immunoprecipitated from cellculture supernatants ( shsy5y cells overexpressing appwt ) . b ) after treatment with 100  nm of a prototypic tripartite bace1 inhibitor ( compound 8  g in ref . 32a ) for 72  h. the a peptides were immunoprecipitated from the conditioned media and analyzed by maldims . the inset shows the signal assigned to a340 ( 2 ) in isotopic resolution , resulting in an accurate mass determination with only 108  ppm relative mass error ( observed mass : [ m+h ] 
 obsd=4685.852 ; calcd . , a240 , and a340 ( 2 ) by cief immunoassay . starting from stock solutions in dmso ( 1  mg  ml ) , the indicated a peptides were prediluted with 20  mm bicine buffer ( ph  7.6 , 0.6  % chaps ) and analyzed by cief immunoassay as described previously.18 the final concentrations of the a peptides in the microcapillaries were 25  ng  ml , each . the immunological detection was achieved with monoclonal antia antibody wo2 ( millipore ) in combination with biotinylated antimouse secondary antibody and streptavidincoupled horseradish peroxidase ( proteinsimple ) . the electropherograms shown ( baselinecorrected signals ) were recorded with 30  s of exposure time . we have developed an experimental procedure for the selective synthesis of the nterminally elongated a peptides a338 ( 1 ) , a340 ( 2 ) , and a342 ( 3 ) . in depth biophysical characterization indicated that all three elongated peptides are prone to aggregate into thioflavin  tpositive amyloid fibrils . the cellular production from app appears to occur independently of bace1 activity in transfected shsy5y cells . given their occurrence in blood , the elongated a peptides starting at val(3 ) may have relevance in the context of biomarker research . it is expected that the synthetic availability of these a variants will advance the characterization of their potential roles under physiological or pathological conditions . experimental procedure for the synthesis of the peptides 13 : the a peptides were prepared by fmoc solidphase peptide synthesis using an abi 433a peptide synthesizer with uvdetector . wang resin ( novapeg wang resin , 0.62  mmol  g , novabiochem ) was used as solid support , hbtu ( 2(1hbenzotriazol1yl)1,1,3,3tetramethyluronium hexafluorophosphate ) as activation agent , and piperidine for deprotection . the resin ( wang linker on polyethylene glycol , 0.1  mmol , novabiochem ) was manually loaded with the first amino acid using msnt ( 1(mesitylene2sulfonyl)3nitro1,2,4triazole ) activation in order to prevent racemization.36 for this purpose , the amino acid ( 20  equiv compared to the resin ) was dissolved in ch2cl2 ( 6  ml ) and thf ( 23  ml ) under an argon atmosphere . 1methylimidazole ( 15  equiv compared to the resin ) and finally msnt ( 20  equiv compared to the resin ) were added . this solution was added to the resin and stirred gently for 4  h. the mixture was transferred to the synthesizer , in which washing with ch2cl2 and nmp was done . then , acetylation of potentially free nterminal groups at the resin was achieved by treatment with ac2o / ipr2net in nmp . for the assembly of the peptide standard cycles were used with the exception of coupling times , which were increased from 15  min to 35  min and for some amino acids to 50  min ( see below ) . for the synthesis of the peptides 13 , a twofold coupling was executed for ser(26 ) and ala(30 ) . for the following amino acids extended coupling times of 50  min were applied : ala(2 ) , glu(3 ) , phe(4 ) , arg(5 ) , his(6 ) , his(14 ) , gln(15 ) , lys(16 ) , leu(17 ) , val(18 ) , phe(19 ) , asp(23 ) , val(24 ) , gly(25 ) , ser(26 ) ( with twofold coupling ) , asn(27 ) , lys(28 ) , gly(29 ) , ala(30 ) ( with twofold coupling ) , ile(31 ) , ile(32 ) , gly(33 ) , and leu(34 ) . after drying in vacuo , the peptide was cleaved from the resin with tfa/1,2ethanedithiol / h2o / ipr3sih ( 95:2:2:1 ) at room temperature for 90  min . for a342 ( 3 ) , some of the peptide already precipitated on treatment with the cleavage reagent . the resin was removed by filtration and the peptide precipitated in meotbu at 78  c . the resulting white solid was collected by twofold centrifugation ( the solution from first centrifugation was centrifuged a second time ) . the peptides were dissolved in h2o with 0.1  % tfa [ for a338 ( 1 ) and a340 ( 2 ) ] or hexafluoroisopropanol [ for a342 ( 3 ) ] and subsequently purified by preparative hplc ( varian prepstar system , with varian prostar model 320 uv and an evaporative lightscattering detector , els 1000 , polymer laboratories ; column : reversed phase c8 , 30250  mm , vydac 208tp1030 ; flow rate : 20  ml  min ) using h2o with 0.1  % tfa as eluent a and mecn with 0.1  % tfa as eluent b. for 1 and 2 , a gradient of 20  % to 45  % of eluent b over a period of 20  min afforded the peptides a338 ( 1 ) ( 25.5  mg , 5.7  mol , 5.7  % ) and a340 ( 2 ) ( 22.1  mg , 4.7  mol , 4.7  % ) as colorless amorphous solids after lyophilization . for 3 , a gradient of 60  % to 90  % of eluent b over a period of 20  min afforded the peptide a342 ( 3 ) ( 16.3  mg , 3.3  mol , 3.3  % ) as colorless amorphous solid after lyophilization . the purity of the peptides was confirmed by analytical hplc ( agilent model 1100 with g1315b uvdad and evaporative lightscattering detector , els 1000 , polymer laboratories ; column : reversed phase c8 , 4.6250  mm , vydac 208tp104 ; flow rate : 1.0  ml  min ) . analysis was performed with h2o with 0.1  % tfa as eluent a and mecn with 0.1  % tfa as eluent b using a gradient of 20  % to 50  % of eluent b over a period of 30  min for a338 ( 1 ) and a340 ( 2 ) . for a342 ( 3 ) , a gradient of 60  % to 90  % of eluent b was used over a period of 30  min . the identity of the products was confirmed by hrms ( ltq orbitrap xl , thermoscientific ) at a resolution of 60  000 and a mass accuracy of 3  ppm ( see supporting information ) . preparation of monomeric a peptide samples : to dissociate preformed aggregates , the a peptide powders were dissolved in 20  mm naoh at 2  mg  ml peptide concentration and sonicated for 1  min.37 after shaking for 30  min at 4  c , the a peptide solutions were exposed to ultracentrifugation ( 100  000  g , 4  c , 1  h ) . subsequently , the supernatants were aliquoted , flashfrozen by liquid nitrogen , and stored at 80  c until use . a142 was purchased from peptide specialty laboratories ( psl ) ( heidelberg , germany ) with a purity of more than 95  % . nmr spectroscopy : nmr measurements were performed on a 600  mhz bruker spectrometer equipped with a cryogenic probe . nmr samples contained 0.25  mg  ml a in 20  mm sodium phosphate ( ph  7.4 ) plus 100  m dss ( 4,4dimethyl4silapentane1sulfonic acid ) for chemical shift referencing ( 0  ppm ) . 2dh , htocsy and noesy spectra were obtained at 5  c using mixing times of 60 and 80  ms for tocsy and 200 and 300  ms for noesy experiments . the pfgnmr experiments were measured at 5  c as described previously.21 translational diffusion coefficients , obtained after gradient calibration,21 were converted to hydrodynamic radii according to the stokes the hydrodynamic radius of dioxane was used for the viscosity correction . for nmrbased monomerconsumption assays , 1dh spectra were measured before and after 24  h of incubation under aggregationprone conditions ( 37  c , gentle stirring ) . the residual a monomer levels were quantified on the basis of nmrsignal intensities in the methyl region using dss as internal intensity reference . for realtime monitoring of a monomer consumption , seeds of aggregation were added to 0.2  mg  ml a samples ( ph  7.4 , buffered with 50  mm sodium phosphate , containing 50  mm nacl and 500  m dss ) , then consecutive 1dh spectra were measured at regular intervals , while the a samples were incubated at 37  c within the nmr spectrometer . the aggregation seeds were prepared after mixing the aggregated samples of a338 ( 1 ) , a140 , a340 ( 2 ) , a142 , and a342 ( 3 ) , spinning down the fibrils ( 100  000  g , 15  c , 4  h ) , and sonication of resuspended a fibrils for 30  min . the concentration ratio of seed to monomeric a was smaller than 1  % . circular dichroism : cd measurements were performed on a j815 jasco spectropolarimeter using a cuvette of 1  mm path length . before and after 24  h of incubation under aggregationprone conditions ( 37  c , gentle stirring ) , the cd spectra of a samples ( 0.2  mg  ml , ph  7.4 buffered with 20  mm sodium phosphate ) were recorded at 20  c between 190 and 260  nm . thioflavin  t ( tht ) aggregation assay : a samples ( 0.2  mg  ml , ph  7.4 buffered with 20  mm sodium phosphate ) were incubated under aggregationprone conditions ( 37  c , gentle stirring ) . before and after the specified time points of incubation , the tht assays were performed by mixing 10  l aliquots of the a samples with 2  ml tht solution ( 5  m , ph  8.0 buffered with 50  mm glycine ) . the excitation wavelength was 446  nm and the tht emission intensities were averaged between 478 and 488  nm . for the kinetic experiments presented in figure  6 , a samples ( 0.04  mg  ml in pbs , containing 25  m tht ) were incubated in mild aggregation conditions ( rt , gentle stirring ) and development of the tht fluorescence was followed in a realtime manner with the excitation and emission wavelengths of 446 and 482  nm and slits of 10  nm . the analysis of each tht trace was performed according to the equation : df / dt = k 
 f  ( f 
 limf).26 , 30 
 
 electron microscopy : a samples ( 0.2  mg  ml , ph  7.4 buffered with 25  mm hepes ) were incubated at 37  c with gentle stirring . after 24  h of incubation , samples were deposited onto carboncoated copper mesh grids and negatively stained with 2  % ( w / v ) uranyl acetate . the samples were examined using a philips cm 120 biotwin transmission electron microscope ( philips inc . eindhoven , the netherlands ) . 
 cell culture , inhibitor treatment and aimmunoprecipitation : cultivation of transfected shsy5y cells overexpressing human wildtype app695 with an aminoterminal myc tag and a carboxyterminal flag tag and treatment with a prototypic tripartite bace1 inhibitor ( compound 8  g in ref . 32a ) were described previously.32a after 72  h of treatment with 100  nm of tripartite inhibitor or 0.1  % dmso ( vehicle control ) , the conditioned media were collected and the a peptides immunoprecipitated as described previously.18 , 32a a volume of 800  l of cellculture supernatant was mixed briefly with 200  l of a 5x ipdetergent buffer ( 250  mm hepes / naoh , ph  7.4 ) , 750  mm nacl , 2.5  % ( v / v ) nonidet p40 ( igepal ca630 ) , 1.25  % ( w / v ) sodium deoxycholate , 0.25  % ( w / v ) sds ) and incubated overnight at 4  c under rotation with 25  l of magnetic beads ( magnetic sheepantimouseigg dynabeads m280 from life technologies , precoated with monoclonal antia antibody 6e10 , covance ) . the immobilized immune complexes were collected on a magnetic stand and washed 25  min with pbs , 25  min with 50  mm nh4hco3 and once with ultrapure h2o . for maldims analysis the immunoprecipitated a peptides were eluted for 5  min in 50  l of 0.1  % hcho/0.05  % noctyl dglucopyranoside ( ogp ) at rt . mass spectrometry : the masses of the intact a peptides were determined as described previously.18 in a vacuum centrifuge , the volume of the eluted fraction from immunoprecipitation was reduced to about 10  l and 0.3  l were spotted onto an anchorchip target ( bruker daltonics , bremen , germany ) precoated with 2cyano4hydroxycinnamic acid . after drying , the samples were washed twice with ( nh4)h2po4 ( 10  mm in 0.1  % tfa ) and analyzed by mass spectrometry using an ultraflex maldi tof / tof instrument ( bruker daltonics , bremen , germany ) as described previously.13 , 18 
 
 isoelectric point determination by the ciefimmunoassay : the isoelectric point ( pi ) of the peptide a340 ( 2 ) was compared to those of a140 and a240 ( ana spec inc . , fremont ca ) by capillary isoelectricfocusing immunoassay ( cief immunoassay ) on a nanopro 1000 device ( protein simple ) according to a reported procedure.18 the immunological detection shown in figure  7 was achieved with monoclonal antia antibody wo2 ( millipore ) with a 1:50 dilution in combination with biotinylated goatantimouse secondary antibody ( 1:100 ) and streptavidincoupled horseradish peroxidase ( 1:100 ) ( protein simple ) . as a service to our authors and readers , this journal provides supporting information supplied by the authors . such materials are peer reviewed and may be reorganized for online delivery , but are not copyedited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors .
abstractin addition to the prototypic amyloid ( a ) peptides a140 and a142 , several a variants differing in their amino and carboxy termini have been described . 
 synthetic availability of an a variant is often the key to study its role under physiological or pathological conditions . 
 herein , we report a protocol for the efficient solidphase peptide synthesis of the nterminally elongated apeptides a338 , a340 , and a342 . 
 biophysical characterization by nmr spectroscopy , cd spectroscopy , an aggregation assay , and electron microscopy revealed that all three peptides were prone to aggregation into amyloid fibrils . 
 immunoprecipitation , followed by mass spectrometry , indicated that a338 and a340 are generated by transfected cells even in the presence of a tripartite site amyloid precursor protein cleaving enzyme  1 ( bace1 ) inhibitor . 
 the elongated a peptides starting at val(3 ) can be separated from nterminallytruncated a forms by highresolution isoelectricfocusing techniques , despite virtually identical isoelectric points . 
 the synthetic a variants and the methods presented here are providing tools to advance our understanding of the potential roles of nterminally elongated a variants in alzheimer 's disease .