urine has now become one of the most attractive biological fluids in clinical proteomics [ 1 , 2 ] .
a number of urinary proteomic studies have been conducted and have revealed urinary biomarker candidates for renal systemic diseases and malignancies of urinary tract [ 35 ] .
proteomic analysis of urines can be applied to biomarker search in nonrenal diseases as well [ 68 ] .
although urinary proteome analyses have been conducted by various gel - based and gel - free techniques , comprehensive urinary proteome analysis is not an easy task because the urine has very diluted protein concentration with high levels of salts .
sample preparation , processing , and storage for urinary proteomics have been reviewed [ 1013 ] .
more recently , an optimized quantitative proteomic strategy for urine biomarker discovery was described . in any event ,
maximal protein recovery from urine is essential for detecting trace quantities of proteins present in urine for potential biomarker discovery . for this purpose ,
one possible factor responsible for loss of trace amounts of urinary proteins could be adsorption to sample tubes , but this issue has not been well addressed so far to our knowledge .
many attempts have been made to prevent the adsorption of plasma proteins and to improve blood compatibility by surface modification [ 15 , 16 ] .
ishihara and coworkers reported on rapid development of hydrophilicity and protein adsorption resistance by polymer surfaces bearing poly(2-methacryloyloxyethyl phosphorylcholine ( mpc)-co - n - butyl methacrylate ) ( poly(mpc - co - bma ) ) [ 17 , 18 ] .
we took advantage of this coating method in the present study and assessed whether conventionally used tubes adsorb trace amount of urinary proteins and , if any , whether the surface coating by poly(mpc - co - bma ) can minimize the adsorption .
poly(mpc - co - bma ) was obtained from ai bio - chips co. , ltd ( tokyo , japan ) .
a total of 6 different types of conventional urine collection tubes were used in this study .
tubes made from polystyrene ( ps ) ( cat # 10200 ) , polypropylene(pp ) cat # 72200 , polyethylene terephthalate(pet ) ( cat # 23540 ) , and styrene - butadiene copolymers / methyl methacrylate - styrene ( sbc / ms ) ( cat # 17300 ) were purchased from toyo kagaku kizai co. , ltd .
tubes made from acrylonitrile - styrene ( as ) copolymers ( cat # 479511373 ) were from nittobo medical co. , ltd .
, japan and those made from styrene - butadiene copolymers ( sbc ) ( cat # 3324a000a-10 ) were from asiakizai co. , ltd .
the conventional tubes made by as were coated by poly(mpc - co - bma ) as described by futamura et al . .  
an aliquot of the samples was taken for routine urinalysis , and the rest of the samples were centrifuged ( 700  g , 5  min at room temperature ) , and the supernatant was subjected to assess protein adsorption on test tubes as described below .
one ml of two different levels of pooled proteinuric urines ( equivalent to 15  mg / dl and 50  mg / dl , resp . ) obtained from 10 patients with renal disease were put into poly(mpc - co - bma)-coated and noncoated urine correction tubes and were kept at room temperature for 15  min .
after aspiration of the urines , the tubes were washed with 200  l of pbs three times . after the third wash and pbs being aspirated , 100  l of page sample buffer ( electrophoresis ) ( 50  mm tris - hcl , ph 6.8 containing 50  mm dithiothreitol , 0.5% sds , and 10% glycerol ) or 1% tca aqueous solution ( maldi - tof ms analysis ) was added and the tubes were vortexed for 30  sec to dissolve possibly adsorbed proteins . the solution which contained proteins possibly adsorbed on the urine tubes was then analyzed using sds - page ( perfect nt gel w , 1020% acrylamide , 20  wells ; drc co. , ltd . ,
the gel was stained with cbb ( phastgel blue r ; ge healthcare , little chalfont , uk ) .
the proteins separated by sds - page were identified by in - gel tryptic digestion of the proteins followed by ms . in - gel
molar quantities of recovered peptide fragments were estimated from the staining intensity of the sds - page bands that were digested in - gel with trypsin .
digested peptides roughly equivalent up to 1  pmol of protein were injected into a trap column : 0.3  5  mm l - trap column ( chemicals evaluation and research institute , saitama , japan ) , and an analytical column : 0.1  50  mm monolith column ( amr , tokyo , japan ) , which was attached to a hplc system ( nanospace si-2 ; shiseido fine chemicals , tokyo , japan ) .
the solvent composition of the mobile phase was programmed to change in 35  min cycles with varying mixing ratios of solvent a ( 2%  v / v ch3cn and 0.1%  v / v hcooh ) to solvent b ( 90%  v / v ch3cn and 0.1%  v / v hcooh ) : 550%  b 20  min , 5095%  b 1  min , 95%  b 3  min , 955%  b 1  min , 5%  b 10  min .
purified peptides were introduced from hplc to an ltq - xl ( thermo scientific , calif , usa ) , an ion trap mass spectrometer ( itms ) , via an attached pico tip ( new objective , mass , usa ) .
the ms and ms / ms peptide spectra were measured in a data - dependent manner according to the manufacturer 's operating specifications .
the mascot search engine ( matrix science , london , uk ) was used to identify proteins from the mass and tandem mass spectra of peptides .
peptide mass data were matched by searching the human international protein index database ( ipi , july 2008 , 72079 entries , european bioinformatics institute ) using the mascot engine .
the minimum criterion of the probability - based mascot / mowse score was set with 5% as the significant threshold level . for 2-de analysis
, we used the method described by oh - ishi et al .   and kawashima et al .
briefly , one ml aliquots of urine samples kept at room temperature for 15  min in poly(mpc - co - bma)-coated and noncoated urine correction tube were concentrated up to 20-fold by bjp concentrator ( prochem , ma , usa ) to 50  l and lyophilized .
the lyophilizate was resuspended by 200  l of immobiline reagent ( 7  m urea , 2  m thiourea , 4% chaps , 2% dtt , 2% pharmalyte , broad range ph 310 ) .
finally , 50  l of the 5-fold urine sample was applied to the ief agarose gel .
the agarose gel was then transferred to the perfect nt gels w ( 1020% gradient of polyacrylamide gel ; drc .
the protein spots were detected , quantified , and matched with the 2-de gel view analysis software , progenesis samespots ( nonlinear dynamics ltd .
the protein spots were excised from the gel and identified , as we previously described .
one ml aliquots of urine samples ( containing 50  mg / ml protein ) were kept at room temperature for 15  min in poly(mpc - co - bma)-coated and noncoated urine collection tube .
proteins possibly adsorbed on the tubes were collected as described above for the gel - based method and were analyzed by the maldi - tof ms . to obtain quantitative data of the possibly adsorbed proteins , we used stable isotope - labeled 5.9  kda fibrinogen alpha c chain fragment ( fic 5.9 ) as an internal standard as described by sogawa et al . .
we obtained the stable isotope - labeled synthetic fic 5.9 from the anygen co. , ltd .
( the underlined amino acids were synthesized with c , n uniformly labeled fmoc amino acids . ) . in urine analysis ,
ten microliters of sid ( stable isotope - labeled ) -fic 5.9 solution ( 0.5  pmol/l sid - fic 5.9 , mb - wcx binding solution ) and 5  l of urine sample were transferred to a 200  l pcr tube ( thermo fisher scientific k.k . ,
ten microliters of sid - fic 5.9 solution ( 0.025  pmol/l sid - fic 5.9 , mb - wcx binding solution ) and 5  l of extracted samples ( urine samples kept in tube ) were transferred to a 200  l pcr tube .
a 10  l homogenous magnetic particle solution was added , mixed with the other solutions , and allowed to sit for 5  min .
the pcr tubes were placed in a magnetic bead separator ( mbs ; bruker daltonics gmbh ) for 30  s for magnetic fixation of the mb - wcx particles .
we added 100  l of the washing solution and carefully mixed it with the magnetic beads .
we then replaced the tube into the mbs device and moved it back and forth between adjacent wells on each side of the device 's magnetic bar .
after fixation of the magnetic beads for 30  s , the supernatant was aspirated .
after the final wash , we eluted the bound molecules by incubating them for 1  min with 5  l mb - wcx elution solution and then used the mbs device to collect the eluate . for the final step
, we added 5  l of the mb - wcx stabilization solution to the eluate .
we then mixed 1  l of the eluate with 5  l of a matrix solution ( 0.3 
g / l a - cyano-4-hydroxycinnamic acid in ethanol : acetone , 2  :  1 ) .
we spotted 1  l of the mixture onto an anchorchip target plate ( bruker daltonics gmbh ) and allowed it to dry .
protein calibration standard ( protein calibration standard 1 , bruker daltonics gmbh ) was dissolved in 125  1  l .
we applied 0.5  1  l of the solution to target spots in proximity to the urine samples for external calibration .
we placed the anchorchip target plate into the autoflex ii tof / tof mass spectrometer ( bruker daltonics gmbh ) and into the ultraflex iii tof / tof mass spectrometer ( bruker daltonics gmbh ) , which is controlled by flexcontrol software 3.0 ( bruker daltonics gmbh ) .
spectra were acquired in a positive linear mode in the mass range of 600 to 10,000  da .
the concentration of the proteins adsorbed to urine collection tube was estimated from the ratio of the peak intensity of adsorbed proteins to the peak intensity of sid - fic 5.9 . for identification of peptides as we previously described ,
the anchorchip target plate was also placed in an ultraflextreme tof / tof mass spectrometer ( bruker daltonics ) and the maldi - tof / tof ms / ms spectrum was recorded in lift mode .
the maldi - tof / tof ms / ms spectrum was subjected to a database search using the mascot ( matrix science , london , uk ) database search engine .
the search parameters were as follows : no enzyme specificity , 25  ppm mass tolerance for the parent mass , and 0.2  da for fragment masses .
one hundred urine samples requested for urinalysis on routine basis at the division of laboratory medicine and clinical genetics , chiba university hospital were used .
the urine samples were aliquoted ( 10  ml ) to poly(mpc - co - bma)-coated and noncoated collection tubes and were kept at room temperature for 15  min before use .
nine different quantitative urinalysis such as protein , glucose , creatinine , microalbumin , beta-2-microglobulin , amylase , n - acetyl--d - glucosaminidase , urea nitrogen , uric acid , and six kinds of electrolytes were conducted using biomajesty jca - bm6010 ( jeol ltd .
ten different parameters are assessed : specific gravity ( sg , measured via a built - in refractometer ) , erythrocytes , leukocytes , nitrite , ph , protein , glucose , ketones , bilirubin , and urobilinogen .
this equipment includes digital imaging and auto - particle recognition ( apr ) ( chatsworth , ca , usa ) software to classify urine particles and quantitatively report results . in this study ,
4 categories red blood cells ( rbc ) , white blood cells ( wbc ) , squamous epithelial cells ( sec ) , and casts were classified by the apr software .
we evaluated the statistical significance using ibm spss statistics 18 software ( spss inc . ,
il , usa ) . a p < 0.05 was considered significant using the mann - whitney u test .
proteins adsorbed on the poly(mpc - co - bma)-coated and noncoated tubes were analyzed by sds - page . as shown in figure 1(a ) , a few distinct protein bands ( 60  kda , 66  kda and 100  kda ) were noted in samples obtained from noncoated as tubes .
no clear bands were visible in samples obtained from poly(mpc - co - bma)-coated as tubes under these experimental conditions .
lc - ms analysis of trypsin digests of these bands identified 11 proteins as listed in table 1 .
protein adsorption on the tubes was observed in 6 different types of conventionally used urine collection tubes as shown in figure 1(b ) .
urine specimens kept in poly(mpc - co - bma)-coated and noncoated tubes were subjected to the agarose 2-de as described in the methods section .
nine protein spots the intensities of which were significantly greater ( p < 0.05 ) in samples kept at poly(mpc - co - bma)-coated tubes compared with those kept at noncoated tubes were selected based on the results obtained in seven different experiments .
these differences were most likely as the results of more protein adsorption on noncoated tubes .
these spots were excised and subjected to in - gel trypsin digestion followed by lc - ms .
proteins and/or peptides adsorbed on the conventional urine tubes were also detectable by the maldi - tof ms .  
the intensities of the two peaks ( 2556  m / z and 2654  m / z ) were notably greater in samples obtained from poly(mpc - co - bma ) noncoated tubes .
similar results were obtained in 7 different experiments ; the expression levels of the two peaks expressed as the ratio to the internal standard , sid - fic 5.9 , were significantly greater ( p < 0.001 ) in poly(mpc - co - bma ) noncoated tubes than in coated tubes . using maldi - tof / tof ms / ms technology , we successfully identified the two peaks ( 2556  m / z and 2654  m / z ) as internal sequences of the fibrinogen alpha c chain fragment .
the peptide sequences of the 2556  m / z and 2654  m / z were deagseadhegthstkrghaksrp and deagseadhegthstkrghaksrpv , respectively .
the mean value of the ratio of m / z 2654 to sid - fic5.9 was 5.70 ( figure 2(a ) right panel ) in poly(mpc - co - bma ) noncoated urine collection tube .
it is 0.85  g / ml when converting it into the protein concentration . 

figure 2(b ) shows representative view of the spectrum of urine samples kept in urine tubes .
the relative intensities of the two peaks ( 2556  m / z and 2654  m / z ) were greater in the samples kept in poly(mpc - co - bma)-coated tubes than those kept in noncoated tubes .
similar results were obtained in 7 other experiments ; the differences quantified using the sid - fic 5.9 were statistically significant ( p < 0.007 for 2556  m / z and p
the quantitative values of urinalysis parameters in samples kept in poly(mpc - co - bma)-coated and noncoated collection tubes are presented in table 3 .
they were all comparable between the two groups in linear regression equation , slope of linearity , correlation coefficients ranged from 0.997 to 1.014 .
the results of dipstick urinalysis and urinary sediment determinations were also comparable between the two groups .
the issue of preanalytical factors affecting sample integrity is often overlooked and yet is critically important . although preanalytical factors for serum or plasma proteome analysis have been extensively studied , the impact of adsorption of proteins and peptides on urine tubes on biomarker discovery using urinary proteomics is not well investigated .
the results of this study indicate that conventionally used urine collection tubes adsorb proteins and/or peptides and that the surface coating of the tubes by poly(mpc - co - bma ) can minimize the adsorption without any significant effects on routine chemical determinations . in this study ,
urine samples were kept in poly(mpc - co - bma)-coated and noncoated tubes for 15  min .
this is because it is generally known that proteins adsorb onto a surface within a few minutes when the material contacts body fluids such as blood , plasma , and tears [ 2426 ] .
protein adsorption is one of the most important phenomena in determination of the biocompatibility of materials [ 16 , 18 ] .
polymers composed of mpc and hydrophobic alkylmethacrylate units have been extensively used in many medical devices as coating materials to improve the blood compatibility of these devices [ 1518 ] . however
, this coating requires a long wetting pretreatment time to achieve equilibrium hydration by the reorientation of the phosphorylcholine groups [ 16 , 27 ] . in this study ,
urinary proteins were found to be adsorbed on poly(mpc - co - bma ) noncoated urine collection tubes made from six different types of materials .
recently , futamura et al .   succeeded in rapid development of hydrophilicity and protein adsorption resistance poly(ethylene terephthalate ) ( pet ) surfaces bearing poly(mpc - co-2-vinylnaphthalene(vn ) ) ( pmvn ) .
it should be considered , however , that coating effects on the plastic tubes appear to be dependent on the initial properties of the plastic tubes .
we took advantage of this coating method and showed that protein adsorption can be reduced in urine samples as well .  
most of the proteins listed in table 1 are representative protein in urine and have theoretical isoelectric points between 4.7 and 7.0 , suggesting that proteins with isoelectric point of this range are likely to be adsorbed on the conventional urine tubes employed in this study . since the ph of the urine samples kept in poly(mpc - co - bma)-coated and noncoated tube was similar , it is unlikely that the differences obtained in this study were due to ph difference .
it has been reported that the factor responsible for protein adsorption to the plastic tube might depend on the relation of sample ph and protein pi .
three proteins ( alpha-1-antitrypsin , igkv1 - 5 protein , prostaglandin - h2 d - isomerase ) were detected in common for two different comparisons .
ceruloplasmin , one of the proteins listed in table 1 , is a biomarker of uranium nephrotoxicity .
the use of the coated tubes did not have any impact on the urine analysis of routine parameters .
since there were no significant differences in the quantitative data of abundant urinary proteins including albumin and beta-2-microglobulin , the effects of adsorption on abundant proteins may be minimal .
but , in searching for urinary protein biomarkers with low concentration , possible adsorption on conventional urine tubes should be considered .
since the material used in the conventional and noncoated tubes employed in the present study is widely used around the world , possible adsorption of trance amount of proteins to urine collection tubes should be considered in proteome analyses of urine samples .
urine is one of the attractive biofluids in clinical proteomics . in chasing very low abundance urinary proteins and peptides ,
however , loss of biomarker candidates by adsorption on urine tubes should be considered . in this study
, we found that protein adsorption on the conventionally used urine collection tubes is not negligible , and that the adsorption can be reduced by using a tube coated by hydrophilic polymers without any effects on routine urinalysis .  
i believe that these findings should be shared by those who are interested in urinary proteomic study .
 one possible factor determining recovery of trace amount of protein biomarker 
                     candidates during proteome analyses could be adsorption on urine tubes . 
 this 
                     issue , however , has not been well addressed so far . 
 recently , a new technical 
                     device of surface coating by poly(2-methacryloyloxyethyl phosphorylcholine 
                         ( mpc)-co - n - butyl methacrylate ( bma ) ) 
                         ( poly(mpc - co - bma ) ) has been developed mainly to prevent the 
                     adsorption of plasma proteins . 
 we assessed whether conventionally used urine 
                     tubes adsorb trace amount of urinary proteins and , if any , whether the surface 
                     coating by poly(mpc - co - bma ) can minimize the adsorption . 
                     
 proteinuric urine samples were kept in poly(mpc - co - bma)-coated 
                     and noncoated urine tubes for 15 min and possibly adsorbed proteins and/or 
                     peptides onto urine tubes were analyzed by sds - page , 2-de , and the maldi - tof ms . 
                     
 it was found that a number of proteins and/or peptides adsorb on the 
                     conventionally used urine tubes and that surface coating by 
                         poly(mpc - co - bma ) can minimize the adsorption without any 
                     significant effects on routine urinalysis test results . 
 although it remains to 
                     be clarified to what extent the protein adsorption can modify the results of 
                     urinary proteome analyses , one has to consider this possible adsorption of 
                     urinary proteins when searching for trace amounts of protein biomarkers in 
                     urine . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Summary