parkinson s disease ( pd ) is one 
 of the most common neurodegenerative 
 disorders , with an estimated prevalence of approximately 1 to 2% among 
 people 65 years of age or older .
clinically , pd patients 
 show motor ( e.g. , tremor and rigidity ) and nonmotor ( e.g. , dementia 
 and depression ) symptoms .
it is believed that 
 motor symptoms are largely attributable to the loss of dopaminergic 
 neurons in the substantia nigra , whereas nonmotor symptoms involve 
 many other brain regions .
effective treatment of pd has been hampered partially due to the 
 fact that even newly diagnosed patients are already at relatively 
 advanced stages pathologically .
the other difficulty is lack of objective 
 assessment of disease progression , which has led to extensive research 
 on biomarkers that can be employed for early diagnoses of pd or assessing 
 its progression . as of today
, the best pd protein biomarkers 
 are those found in 
 cerebrospinal fluid ( csf ) , the body fluid that is in direct 
 contact with brain and spinal cord .
discovery 
 of peripheral biomarkers reflecting motor components of pd is highly 
 desirable because csf - based tests depend on the comparatively more 
 invasive process of lumbar puncture that requires more specialized 
 training to collect samples and are therefore not acceptable by some 
 subjects , particularly those without apparent clinical symptoms .
however , 
 peripheral biomarker discovery has been largely unsuccessful , due 
 primarily to factors intrinsic to blood ; specifically , the plasma 
 or serum proteome is quite complex , proteins are present across a 
 wide dynamic range , and target proteins are often of extraordinarily 
 low abundance .
thus , accurately detecting 
 and quantifying brain - derived or disease - specific proteins in blood 
 is challenging for current technologies .
that said , 
 a few blood biomarkers discovered recently , for example , egf and apoa1 , 
 appear to be related to cognitive impairment or disease onset in pd .
additionally , it has been suggested recently that -synuclein 
 pathology , a key component of pd pathogenesis , exists in peripheral 
 nerves , leading to a report demonstrating 
 abnormal autonomic nerve staining in skin biopsies in pd . to facilitate peripheral biomarker discovery , 
 in the current investigation
, 
 we began with proteins identified in our previous proteomic investigations 
 using human brain tissues obtained at autopsy and csf obtained from 
 living patients , with a focus on glycosylated proteins , which are highly enriched in body fluids , including 
 plasma .
we hypothesized that some of the brain or csf -derived proteins 
 will reach plasma via mechanisms yet to be defined and that a subset 
 of these proteins or peptides will be detectable in plasma using sufficiently 
 sensitive measurements . to study the presumably low - abundance 
 brain - derived proteins in 
 plasma
, we turned to a current quantitative mass spectrometry ( ms ) 
 technique , selected reaction monitoring ( srm ) , which has emerged as 
 an alternative to immunoaffinity - based measurements of defined protein 
 sets .
srm has the benefit of fast and cost - efficient assay 
 development , and protein quantification by srm in complex samples 
 using predefined assay coordinates is reproducible across different 
 laboratories and instrument platforms .
however , the main advantage of srm is the capacity to quantify multiple 
 proteins in parallel at a low limit of detection and high accuracy . 

it has been reported that srm has the ability to detect plasma proteins 
 at g / ml levels without any sample enrichment or fractionation , which suggests that a further enrichment would 
 be necessary to detect central nervous system ( cns)-derived proteins 
 at lower levels . in recent years , a multitude of enrichment 
 approaches have been 
 developed . among them
, immune affinity depletion can be used to remove 
 the most abundant proteins , improving detection of low - abundance proteins . 
 however , one risk associated with this kind of predepletion is the 
 proteins of interest might also be partially removed .
in contrast , 
 specific peptides of interest can be enriched after digestion ( immuno - srm ) , 
 dramatically increasing their relative concentration ; however , such 
 techniques are limited by the need for highly specific and high - affinity 
 antibodies , which are either not available for all targets or expensive 
 to generate .
we chose to use the n - glycocapture technique , 
 which is an antibody - free , hydrazide - based approach to selectively 
 enrich n - glycopeptides .
additionally , 
 glycosylation is known to be important in pd , and glycoproteins , which are prevalent in extracellular surface 
 proteins and secreted proteins , are ideal sources of biomarkers . therefore , to test our hypothesis in this study , n - glycoproteins were captured from plasma of a larger cohort of pd 
 patients , along with healthy and diseased controls , by a hydrazide - based 
 solid - phase capturing approach , followed 
 by quantification of peptides that are uniquely associated with pd 
 diagnosis or severity based on our previous experimentations .
a total of 282 subjects , including patients with 
 pd or alzheimer s disease ( ad ) and age - matched controls , recruited 
 at the veterans affairs puget sound health care system / university 
 of washington school of medicine , the oregon health and science university , 
 and the university of california at san diego , were included in the 
 investigation .
the subjects consisted of two subcohorts : those collected 
 prior to 2011 ( 75 pd , 15 ad , and 30 controls ) and those collected 
 more recently ( 98 pd , 15 ad , and 49 controls ) .
all plasma samples 
 were obtained after informed consent from patients , and all patients 
 underwent medical history evaluation , physical , and neurological examinations , 
 laboratory tests , and neuropsychological assessments .
all pd subjects 
 met the uk pd society brain bank clinical diagnostic criteria for 
 pd , while ad cases were diagnosed according 
 to nia criteria .
pd patient samples were 
 further categorized based on updrs part iii on - state motor scores 
 to approximate disease stage .
patients with updrs scores < 15 were 
 defined as early - stage pd , those with scores ranging from 1530 
 were classified as midstage pd , while those with scores > 30 were 
 classified 
 as late - stage pd patients .
control subjects were community volunteers 
 in good health and had no signs or symptoms of cognitive impairment 
 or neurological disease ; all control subjects had a mini mental status 
 examination ( mmse ) score between 28 and 30 , a clinical dementia rating 
 ( cdr ) score of 0 , and new york university paragraph recall scores 
 ( immediate and delayed ) of > 6 .
all samples were collected 
 and processed following standard clinical protocols and quality - control 
 procedures at all participating sites , as defined previously .
isolation of n - linked glycopeptides ( n - glycopeptides ) 
 from plasma was performed as previously described . in brief , 
 starting with aliquots of 25 l individual or pooled 
 plasma , samples were diluted 10-fold with ammonium bicarbonate ( 100 
 mm ) , then denatured with 50% tfe ( 2,2,2-trifluoroethanol , j.t .
baker , 
 philipsburg , nj ) and digested with mass - spectrometry - grade trypsin 
 ( promega , madison , wi ) .
the peptides were desalted using c18 cartridges 
 ( waters , milford , ma ) and were then oxidized with 10 mm naio4 .
the resulting oxidized glycopeptides were coupled to hydrazide 
 resin ( affi - prep , bio - rad , hercules , ca ) by incubation in coupling 
 buffer ( 100 mm sodium acetate and 1.5 m sodium chloride , ph 4.5 ) overnight 
 at room temperature with bottom - over - head rotation .
the unbound nonglycosylated 
 peptides were removed by several washes of sodium chloride ( 1.5 m ) , 
 80% acn , and ammonium bicarbonate ( 100 mm ) , respectively .
n - glycopeptides were finally eluted from the resin by the 
 addition of pngase f in 50 mm ammonium bicarbonate , ph 7.5 , and incubation 
 overnight at 37 c . an mcx ( mixed - mode cation exchange ) desalting 
 step using an oasis elusion plate ( waters , milford , ma ) was 
 performed before lc 
glycoproteomes of brain tissue and csf samples from 
 pd and ad patients and age - matched controls were previously investigated .
all n - glycopeptides with quantitative 
 alterations in brain or csf were selected in the initial candidate 
 library . to evaluate the utility of these glycoproteins as diagnostic 
 markers for pd , we tested the 133 srm assay feasible glycopeptides 
 ( derived from 73 n - linked glycoproteins ) in the initial 
 pilot study using a few pooled plasma samples from pd and controls . 

peptide selection criteria include : ( 1 ) length of 820 amino 
 acid residues ; ( 2 ) no chemically unstable residues ( e.g. , ng , dg , 
 qg , n - terminal n , and n - terminal q ) ; ( 3 ) fully tryptic ; ( 4 ) avoiding 
 cysteine residue if possible ; and ( 5 ) sequence specific for the target 
 protein ( i.e. , proteotypic peptides ) .
all peptides used in this study 
 were evaluated using blat ( http://genome.ucsc.edu ) and 
 protein blast ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) 
 searches to ensure uniqueness of the target proteins at both proteomic 
 and genomic levels .
finally , an srm theoretical collision calculator 
 tool ( http://proteomicsresource.washington.edu/cgi-bin/srmcalc.cgi ) was applied to confirm the uniqueness of every q1/q3 pair for the 
 target peptides .
all srm analyses were performed on 
 a tsq vantage triple quadrupole ( qqq ) mass spectrometer ( thermo scientific ) 
 at the university of washington proteomics resource .
the mass spectrometer 
 was coupled to a nanoelectrospray ionization source and a waters nanoacquity 
 uplc system .
glycopeptides from 1 l of original plasma ( 1 
 g peptides ) were loaded onto a c18 trap column ( 20 mm long , 
 75 m i d ) and separated by a 150 mm c18 column ( 75 m 
 i d ) over a 60 min 235% linear acetonitrile gradient .
spray 
 voltage was set at 1700 v. scheduled srm was performed with 5 min 
 retention time windows for most of the peptides and an instrument 
 cycle time of 2000  500 ms .
dwell times were varied depending 
 on the number of concurrent transitions ; in all cases they were at 
 least 10 ms .
both unpurified 
 ( thermo - fisher scientific , germany ) and purified ( aqua - grade , sigma - aldrich , 
 usa ) peptide standards that correspond to natural counterparts (  light  
 peptides ) were synthesized with heavy isotopic lysine ( c6n2 ) or arginine ( c6n4 ) at the c - termini (  heavy  
 peptides ) .
collision energies ( ces ) were determined using the default 
 formula from thermo ( 0.034  precursor mass m / z + 3.3140 ) and then optimized with four additional 
 ce steps ( 5 v , 10 v ) .
the top four abundant transition 
 ( q1/q3 ) pairs , including 4  light  and 4  heavy  

determine 
 the limits of detection ( lod ) and quantification ( loq ) for each target 
 glycopeptide , we titrated heavy peptides at seven concentration points 
 in a reference glycocaptured plasma matrix .
a linear regression algorithm was used for fitting the seven serial 
 dilution data points for each curve .
the endogenous peptides were 
 also monitored in these assays to help determine the amount of each 
 peptide standard to spike - in .
all raw srm data were processed using 
 the skyline targeted proteomics environment ( v1.3 ) ( mccoss lab , university 
 of washington ) software developed for srm data sets .
settings including 0.055 th match tolerance m / z , default peak integration , and savitzky 
peptides 
 with signal - to - noise ratio of at least 3 were considered detectable . 

information including peak area and area ratio of light / heavy peptide 
 pair were output from skyline to a text - delimited format worksheet .
it should also be noted that the glycopeptide capturing method 
 used in the current study has been well - optimized and widely employed , with cvs typically controlled 
 in 1520% range . to further control 
 this variable , in this study
, we included one to two reference plasma 
 samples in every batch of sample preparations as inter- and intrabatch 
 controls .
these samples showed an average of 16.5 and 14% for preanalytical 
 ( capture ) and analytical ( srm ) variation ( cv ) , respectively ; that 
 is , the variations associated with the capture and srm stages were 
 reasonable in our investigation .
the key challenge for the present 
 analysis is a high dimension of the feature vector ( large number of 
 potentially predictive proteins ) versus size of samples within the 
 data set .
missing data were handled using k - nearest neighbor imputation algorithms ( k = 10 ) . repeated ( duplicate ) measurements for the same protein 
all other analyses were generated in prism 6.0 ( graphpad software , 
 la jolla , ca ) or spss 18.0 ( ibm , chicago , il ) .
one - way analysis of 
 variance ( anova ) , followed by the tukey s hsd post hoc test , 
 was used to compare differences between groups .
receiver operating 
 characteristic ( roc ) curves were used to calculate the relationship 
 between sensitivity and specificity for pd versus the healthy control 
 group and hence to evaluate the diagnostic performance of the analytes , 
 either individually or in combinations .
logistic regression was used 
 to determine the best linear combination of peptide analytes for predicting 
 disease status ( versus healthy controls ) , followed by roc analysis 
 on the linear combination .
cutoff value 
 from a roc curve is determined when the sum of sensitivity and specificity 
 is maximal .
additionally , relationships between the analytes and the 
 unified parkinson s disease rating scale ( updrs ) were analyzed 
 with bivariate correlation using pearson s correlation coefficients . 

stepwise multiple linear regression analysis was used to screen for 
 the best predictors ( linear combination of peptide analytes ) that 
 correlate the disease severity ( updrs )
to enrich secreted proteins 
 in body fluids , in the past few years , we profiled n - linked glycoproteins in the brain and csf of patients with pd as 
 compared with age - matched healthy and diseased controls ( patients 
 with ad ) . a subset of proteins has been previously published .
drawing from our previous work , we identified 
 candidate peptides with quantitative alterations ( defined by 50% 
 over control cases ; full list not shown ) and selected a total of 133 n - glycopeptides , representing 73 n - linked 
 glycoproteins based on peptide characteristics , especially amino acid 
 sequence , suitable for srm assays , as defined in the methods section . with the aid of synthetic heavy - isotopic 
 labeled peptides
, srm conditions including q1/q3 transitions , collision 
 energy ( ce ) , retention time ( rt ) , and the spiked - in amount of heavy 
 peptide , were first optimized with a set of pooled samples obtained 
 from healthy controls and pd patients . on the basis of the detectability 
 of target peptides in the pooled samples ( either control or pd ) , 50 
 formerly n - glycosylated peptides ( deamidation happens 
 during glycocapture , the n - glycopeptides monitored 
 in srm are not glycosylated anymore ) derived from 40 glycoproteins 
 ( supplemental table 1 in the supporting information , along with reported biological functions ) were selected as srm 
 targets for further analysis , with spiked synthetic corresponding 
 heavy peptides in the initial stage of the validation to be discussed 
 later .
brief workflow outlining the pipeline for screening 
 plasma pd glycoprotein 
 biomarkers by srm . in the pilot study ,
a primary synthetic peptide 
 library containing 133 unpurified , deamidated n - glycopeptides 
 was used to generate srm assays and test the detectability of these 
 formerly n - glycosylated peptides in glycocaptured 
 plasma .
refined srm assays with optimized settings including retention 
 time ( rt ) were applied in next stages in the pipeline . in initial 
 validation , 50
n - glycopeptides that could be detected 
 in the pilot study were measured in a cohort including 15 pd , ad , 
 and control samples pooled from 75 pd , 15 ad , and 30 control individuals , 
 respectively . the final srm assay library containing 12 purified , 
 deamidated n - glycopeptides
was then used to detect 
 the candidate biomarkers in an independent validation cohort for roc 
 calculation and assessment of disease severity association ( n = 162 ) .
having identified peptides that 
 are reliably detectable in plasma , we then aimed to narrow down the 
 panel of potential biomarker candidates to those showing the largest 
 alterations under the disease conditions .
a total of 120 cases from 
 pd , ad , and age - matched controls were included in this study , with 
 the samples pooled based on disease status . to allow approximation 
 of disease stage correlations , we further split plasma from pd subjects 
 into three subgroups according to updrs - defined disease severity . 
 to reduce the within - pool heterogeneity and facilitate statistical 
 analysis
, we combined samples in each group into three small pools 
 ( control : n = 3 , early pd [ updrs < 15 ] : n = 3 , intermediate pd [ updrs 1530 ] : n = 3 , late pd [ updrs > 30 ] : n = 3 , and ad : n = 3 ) . each pooled sample consisted of 10 individuals with 
 age and gender
evenly distributed , except the ad and early pd groups , 
 where plasma from only five individuals was used because of limited 
 samples in these subcohorts ( table 1 ) .
quantitative 
 assessment of 15 pooled samples revealed clear differences among different 
 diagnostic groups or between different stages of the disease ( supplemental 
 table 2 in the supporting information ) . 

figure 2 shows the hierarchical clustering 
 analysis , demonstrating a clear separation of pd with updrs > 15 
 ( right 
 side , pd2 _ * and pd3 _ * columns ) from the three controls ( left side 
 columns ) .
interestingly , this phenomenon is missing in the three ad 
 pools as well , suggesting that these elevations are unique in pd with 
 updrs > 15 . the three updrs < 15 samples ( pd1_1 , pd1_2 , and pd1_3 ) 
 did not present obvious changes compared with the controls , indicating 
 that the current set of glycoproteins may lack the ability to distinguish 
 pd in the early stage from controls , at least in these pooled samples . 

overall , a total of 12 n - glycopeptides derived from 
 11 glycoproteins were detectable , with more than 50% difference between 
 pd versus controls or between the stages of disease .
consequently , 
 these 12 n - glycopeptides were selected to be the 
 targets in the next stage of validation experiments .
heat map of hierarchical 
 clustering analysis of the 50 n - glycopeptides that 
 were detected in the initial validation 
 cohort containing 15 pooled control , ad , and pd plasma samples .
the 
 analysis was based on srm measured peptide relative abundance against 
 average of three pooled controls and conducted using average linkage 
 and euclidean distance .
pd1 , pd2 , and pd3 represent pd at early , middle , 
 and late stages of the disease , respectively , based on updrs .
( also 
 see table 1 . ) pooled samples , 
 even with multiple pools as performed in our initial validation previously 
 discussed , do not provide precise sensitivity and specificity information . 

accordingly , in the final validation stage ( figure 1 ) , a total of 162 individual plasma samples from an independent 
 cohort of subjects recruited from the same medical center were investigated . 

additionally , to achieve higher - level accuracy , we used aqua level 
 purified heavy isotopic - labeled peptides , with srm running conditions 
 further optimized for the new peptide standards ( supplemental table 
 3 in the supporting information ) .
peptide 
 sequences , estimated plasma concentrations , and limits of detection 
 and quantification of these 12 peptides are presented in table 2 . of note ,
calibration curves were generated in 
 a reference glycocaptured plasma matrix to fully characterize the 
 performances of srm assays .
calibration curves for each aqua peptide 
 are plotted on linear scales in supplemental figure 1 in the supporting information .
representative srm chromatograms 
 of the 12 glycopeptides ( both endogenous and spiked - in standard ) are 
 shown in supplemental figure 2 in the supporting 
 information . with the exception of peptide sema4d - aadytsslnlpdk , 

the remaining 11 peptides were detected in more than 50% of the individuals 
 ( supplemental table 4 in the supporting information ) .
p values and posthoc comparisons between each 
 diagnostic group and age - matched controls were calculated for each 
 of the 12 srm peptides using one - way anova , followed by tukey s 
 hsd post hoc tests ( table 3 ) .
three of the 
 12 n - glycopeptides , derived from glycoproteins prnp 
 ( prion protein ) , golm1 ( golgi membrane protein 1 ) , and ncam1 ( neural 
 cell adhesion molecule 1 ) , were significantly altered between pd versus 
 age - matched controls ( age 50 ; pd , n = 96 ; 
 control , n = 34 ; p < 0.05 ) . among 
 them ,
the plasma concentration of peptide ncam1 was also significantly 
 increased in ad patients compared with age - matched controls ( p < 0.05 ) , suggesting its change may be related to neurodegeneration 
 but not specific to pd .
furthermore , levels of 5 n - glycopeptides ( chl1_iip , chl1_isg , golm1 , hspg2 , and tnc ) were lower 
 in younger controls ( age < 50 ) , indicating that the blood level 
 of these glycoproteins was likely associated with aging processes .
notes : n , deamidated 
 asparagine residues . the 
 limit of detection ( lod ) of 
 each peptide was obtained from the average of lowest concentration 
 point at which all three or four transitions were confidently detected . 

the limit of quantification ( loq ) was obtained from the average of 
 lowest concentration point at which the intensity of the most abundant 
 transition is on the linear scale along with the calibration curves . 

settings in tukey s hsd post hoc tests : 
 alpha = 0.05 ; n = average sample numbers in the two 
 comparison groups .
proteins and peptides in bold are those with statistical 
 alterations unique to pd ( vs age - matched controls and ad ) . significance level : p < 0.01 . significance 
 level : p < 0.05 . on the basis of the performance of each individual 
 peptide on the 
 group difference
, a logistic regression - based multivariate analysis 
 was performed to evaluate the sensitivity and specificity of the three 
 pd unique peptides , alone or in combination with the rest of the 12 
 peptides for discriminating of pd from healthy controls ( age 50 ) 
 ( table 4 ) . as shown in supplemental figure 
 3 in the supporting information ,
the best 
 individual performing peptide was prnp ( area under curve ( auc ) : 0.648 ; 
 sensitivity : 76.8% ; and specificity : 47.1% ) .
the performance of roc 
 became progressively better when more peptides were added to the panel ; 
 a combination of four peptides ( prnp , hspg2 , megf8 , and ncam1 ) improved 
 auc to 0.753 , sensitivity to 90.4% , and specificity to 50.0% ( figure 3a ) .
notably , however , adding more peptides only 
 enhanced roc performance slightly ; for example , a panel of 10 peptides 
 ( prnp , hspg2 , megf8 , ncam1 , icam1 , tnc , golm1 , chl1_iip , chl1_isg , 
 and lsamp ) achieved the following values : auc , 0.840 ; sensitivity , 
 71.7% ; and specificity , 83.3% .
also , as expected , there were more 
 cases having missing values ( i.e. , peptides not detectable ) when more 
 peptides were included ; specifically , the number of missing values 
 for prnp , 4-peptide combination , and 10-peptide combination were 1 , 
 4 , and 53 , respectively . logistic regression of a panel of four n - glycopeptides 
 and correlation with pd progression .
( a ) receiver operating characteristic 
 ( roc ) curve and corresponding area under the curve ( auc ) of the classifier 
 in distinguishing pd from controls are presented .
this classifier 
 includes four formerly n - linked glycopeptides ( prnp , 
 hspg2 , megf8 , and ncam1 ) .
( b ) combination of two n - glycopeptides ( icam 
 1 and megf8 ) correlates with pd progression .
. significant differences 
 among pd subgroups at different disease stages ( updrs < 15 , updrs 
 1530 , updrs > 30 ) and age - matched controls were also observed 
 ( table 3 ) .
next , the correlation of pd severity 
 ( as determined by updrs ) with plasma levels of single peptide or a 
 combination of peptides was evaluated by pearson s correlation . 

a stepwise multiple linear regression analysis was used to screen 
 for the best predictors ( linear combination of peptide analytes ) for 
 disease severity .
although none of the single peptides significantly 
 correlated with updrs , when peptides were considered together , a combination 
 of megf8 and icam1 was identified to correlate significantly with 
 updrs ( figure 3b ; r = 0.293 , p = 0.004 ) .
it has been exceptionally 
 challenging to detect peripheral markers 
 unique to cns diseases , including pd , largely because human blood 
 is extremely complex , with proteins contributed from many organ systems , 
 and cns - derived proteins are exceedingly low in concentration . in 
 this study
, we took a targeted approach , using previously identified 
 cns - related proteins with changes specific to pd as the starting point . 

several objectives are achieved in this study , including ( 1 ) detection 
 of 11 cns - related glycoproteins ( related to pd diagnosis or severity 
 in previous proteomics profiling ) in plasma , a much less invasive 
 sample source ( than brain , csf , or skin biopsy ) , ( 2 ) a combination 
 of several peptides , that is , prnp , hspg2 , megf8 , and ncam1 , provided 
 good diagnostic sensitivity ( 90.4% ) and specificity ( 50.0% ) between 
 pd and controls , and ( 3 ) a combination of two peptides , megf8 and 
 icam1 , significantly correlated with pd severity , as measured by updrs .
all 12 of the n - glycopeptides studied in the final 
 validation study are either relatively specific to the cns or have 
 functions potentially important to pd pathogenesis or cns diseases 
 in general
. however , those that provided good sensitivity and specificity , 
 alone and in combination , warrant further discussion .
prnp ( prion 
 protein or prp ) is most predominantly expressed in the nervous system 
 but occurs in many other tissues throughout the body .
brain barrier ( bbb ) and , when aggregated , becomes a major contributor 
 to a variety of cognitive deficiencies and neurodegenerative diseases , 
 especially creutzfeldt  jakob disease .
prp has been implicated as a receptor and binding partner for a 
 oligomers in ad , and its discovery as a protein 
 altered in pd suggests interesting potential for a role in pd pathogenesis 
 as well .
neural cell adhesion molecule 1 ( ncam1 ) plays important roles 
 in the inflammatory mechanisms associated with neurodegeneration and 
 participates in the neuroprotective response in neurodegeneration .
its up - regulation in pd cases could be a compensatory 
 mechanism during the disease process .
similarly , heparan sulfate proteoglycan 
 2 ( hspg2 ) is an extracellular matrix protein that is primarily synthesized 
 by vascular endothelial and smooth muscle cells , with several proposed 
 functions , including maintenance of the integrity of the bbb , which has been reported to be compromised in 
 pd .
finally , very little is known about 
 megf8 ( multiple epidermal growth factor - like domains 8) and its role 
 in either pd or any other neurodegenerative disorder .
interestingly , 
 a recent study has suggested that this protein could potentially be 
 involved in phagocytic function of astrocytes .
it should be stressed that while the four - peptide 
 panel probably 
 does not reach sufficient ( 80% ) sensitivity / specificity to 
 be used as a sole diagnostic criterion , it performed similarly to 
 the best biomarkers discovered thus far .
for example , in a previous 
 study of -synuclein and dj-1 in csf of pd patients , sensitivity 
 and specificity for distinguishing control from pd were 94 and 50% 
 for dj-1 and 93 and 39% for -synuclein , respectively .
this result is especially notable when considering 
 that the previous results were obtained in csf , a biological fluid 
 in direct contact with the extracellular space in the brain , while 
 the current study used a peripheral fluid .
thus , while the current 
 test performed similarly , it provides at least a promising lead to 
 use a less invasive sample by sensitive detection of cns - related proteins 
 in blood samples .
that said , even a test without ideal sensitivity 
 or specificity could be useful in clinical and research settings ( e.g. , 
 selecting patients for clinical trials or screening for preclinical / premotor 
 patients ) , where they may be useful for screening subjects or patients 
 whose diagnosis would then be confirmed by more expensive or more 
 invasive tests
. of note is also the observation that another 
 glycoprotein , golm1 
 ( golgi membrane protein , also known as golgi phosphoprotein 2 or golgi 
 membrane protein gp73 ) , was significantly changed between pd and controls 
 but did not perform well in logistic regression screening for roc 
 performance .
however , this does not necessarily mean that it is not 
 important to pd pathogenesis or development .
indeed , the golgi complex , 
 including gp73 , plays a key role in the sorting and modification of 
 proteins exported from the endoplasmic reticulum , which has been demonstrated 
 to be dysfunctional in pd or pd models . to date , biomarkers that could robustly correlate with pd 
 severity 
 or progression ( assessed in longitudinal collected samples ) are quite 
 rare , if there are any .
in fact , we are unaware of any 
 such pd candidate markers ( or ad for that matter ) that have been validated 
 by independent studies . to this end ,
our candidate markers , although 
 vetted through discovery and validation phases , require yet another 
 round of validation by independent groups , particularly for the identified 
 models / combinations .
that said , two candidate peptides , megf8 and 
 icam1 , were associated with pd severity in this study .
as previously 
 indicated , there is no clear evidence that megf8 is involved in cns 
 disease at this point , and its potential role should be investigated , 
 particularly if these findings are validated , as it may be a novel 
 pd - related protein .
intercellular adhesion molecule 1 ( icam1 ) is a 
 cell - surface glycoprotein that is typically expressed on endothelial 
 cells and cells of the immune system .
one of the proposed cns functions of icam1 is involvement in vasoconstriction 
 associated with subarachnoid hemorrhage , indicating its role in endothelial dysfunction .
in addition to regulating 
 endothelial functions , icam1 is also a mediator of cellular inflammation 
 and reported to be regulated by dj-1 and -synuclein , two proteins 
 intimately involved in pd , during neurodegeneration .
the increase in circulating icam1 as a function of increasing pd 
 severity is in line with the argument that there is persistent inflammation 
 during pd development and progression .
it remains 
 to be determined , though , whether plasma icam1 is originated from 
 the cns or the other way around .
the two - peptide panel that 
 correlated with disease severity may 
 also be useful in developing an assay to track the progression of 
 pd .
however , as in all biomarker studies , the biomarkers studied here 
 must be further validated in future studies .
moreover , because of 
 the unbiased nature of the discovery stage of this study , some of 
 the peptides discovered here ( including the pair correlating with 
 updrs ) have not previously been associated with pd and are therefore 
 also new putative candidates for future studies of pd pathogenesis .
interestingly , most of these peptides showed a pattern of greatest 
 increase in the midstage pd group , followed by a decrease in the late 
 pd group .
the reasons for this pattern are not yet clear , but it should 
 be emphasized that updrs does not reflect a linear pattern of degeneration 
 in specific brain regions but rather a combination of the neurodegenerative 
 and compensatory processes occurring throughout the brain . moreover , 
 pd processes are highly heterogeneous , and individual disease courses 
 vary substantially .
thus , biomarker patterns may similarly be nonlinear 
 and require interpretation depending on disease stage ( e.g. , screening 
 tests at early stages vs tests for following progression at more advanced 
 stages ) .
it is also notable that although several peptides showed 
 significant 
 differences between control and pd subjects no single peptide correlated 
 with pd severity .
this result resembles those observed for -synuclein , 
 which is reduced in the csf of pd patients but does not correlate 
 with disease severity .
for example , some biomarkers show 
 floor / ceiling effects , in which the maximal change occurs early in 
 the disease and therefore no correlation is observed with further 
 progression .
furthermore , proteins involved in the disease process 
 may undergo nonlinear changes , such as early compensatory up - regulation , 
 followed by decreases accompanying more severe neurodegeneration . 

moreover , it must be considered that the current results were obtained 
 in plasma , while the proteins measured may have originated in the 
 cns .
therefore , alterations in peripheral clearance or mechanisms 
 of transfer across the bbb may change during the course of the disease 
 as well , so plasma levels reflect a complex combination of disease 
 processes .
in 
 the original selection of candidate peptides , we included peptides 
 that changed 50% compared with controls . while this criterion 
 is lenient , the low threshold could be justified in the preliminary 
 steps , where the goal was choosing a pool of candidates for further 
 analysis .
because further elimination of candidates would occur in 
 following steps , to narrow a large number of detectable peptides to 
 a feasible number of promising candidates for srm , failing to identify 
 a candidate was more problematic than including a peptide that may 
 not really change .
an additional limitation was the pooling of subjects 
 used for the first test set . in the preliminary validation step , the 
 primary goal was to identify which of the candidate peptides were 
 most altered in the disease conditions .
we followed our previous strategy 
 of combining samples into small pools by disease status .
this represents a compromise , limiting costs 
 by decreasing the samples to be processed and analyzed but also allowing 
 statistical analyses .
this strategy does have statistical costs , most 
 notably dramatic reduction of the power to detect subtle changes ( due 
 to the reduction from the number of subjects to the number of pools ) 
 and masking the true variability of individual subjects .
however , 
 this limitation can be justified in the context of a preliminary study , 
 in which the goal was to rank peptide candidates by the magnitude 
 of their changes .
that is , while we may not have detected subtle differences 
 between groups in some peptides , these differences may not be the 
 changes of the greatest relevance .
finally , an important aspect 
 of the current study is the increase 
 in efficiency of the use of large proteomics data sets . in biomarker 

research , unbiased studies can produce very large pools of candidate 
 biomarkers , but validation of these candidates is hampered by limitations 
 in assay development for individual proteins .
thus , this strategy 
 of prioritizing peptides that change in the cns under the disease 
 condition , while moving to peripheral fluids and using sensitive , 
 relatively high - throughput assays such as srm , may allow improved 
 use of these data in the future . in conclusion , using a relatively 
 large cohort totaling 282 subjects , 
 several cns - related protein markers , implicating potential novel mechanisms 
 in pd pathogenesis were readily detected in human plasma .
several 
 of them provided good diagnostic sensitivity / specificity for pd or 
 correlated with pd severity .
these results , if validated in independent 
 investigations , could potentially help with establishing cns specific 
 markers in blood for early disease diagnosis , monitoring disease progression 
 or assessment of treatment effects .
 despite 
 extensive research , an unmet need remains for protein biomarkers 
 of parkinson s disease ( pd ) in peripheral body fluids , especially 
 blood , which is easily accessible clinically . 
 the discovery of such 
 biomarkers is challenging , however , due to the enormous complexity 
 and huge dynamic range of human blood proteins , which are derived 
 from nearly all organ systems , with those originating specifically 
 from the central nervous system ( cns ) being exceptionally low in abundance . 
 in this investigation of a relatively large cohort ( 300 subjects ) , 
 
 selected reaction monitoring ( srm ) assays ( a targeted approach ) were 
 used to probe plasma peptides derived from glycoproteins previously 
 found to be altered in the cns based on pd diagnosis or severity . 
 
 next , the detected peptides were interrogated for their diagnostic 
 sensitivity and specificity as well as the correlation with pd severity , 
 as determined by the unified parkinson s disease rating scale 
 ( updrs ) . 
 the results revealed that 12 of the 50 candidate glycopeptides 
 were reliably and consistently identified in plasma samples , with 
 three of them displaying significant differences among diagnostic 
 groups . a combination of four peptides ( derived from prnp , hspg2 , 
 megf8 , and ncam1 ) provided an overall area under curve ( auc ) of 0.753 
 ( sensitivity : 90.4% ; specificity : 50.0% ) . 
 additionally , combining 
 two peptides ( derived from megf8 and icam1 ) yielded significant correlation 
 with pd severity , that is , updrs ( r = 0.293 , p = 0.004 ) . 
 the significance of these results is at least 
 two - fold : ( 1 ) it is possible to use a targeted approach to identify 
 otherwise very difficult to detect cns related biomarkers in peripheral 
 blood and ( 2 ) the novel biomarkers , if validated in independent cohorts , 
 can be employed to assist with clinical diagnosis of pd as well as 
 monitoring disease progression . 