kiernan et al_2002_journal of proteome research_comparative urine phenotyping.pdf

892019 Kiernan et al_2002_Journal of proteome research_Comparative urine phenotypingpdf

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Comparative Urine Protein Phenotyping Using Mass SpectrometricImmunoassay

Urban A Kiernandagger Kemmons A Tubbsdagger Dobrin Nedelkovdagger Eric E Niederkoflerdagger

Elizabeth McConnellDagger and Randall W Nelsondagger

Intrinsic Bioprobes Inc 625 South Smith Road Suite 22 Tempe Arizona 85281 and Arizona State UniversityTempe Arizona 85287

Received October 1 2002

Reported here human urine samples were analyzed for -2-microglobulin ( 2m) transthyretin (TTR)

cystatin C urine protein 1 (UP1) retinol binding protein (RBP) albumin transferrin and human

neutrophil defensin peptides (HNP) using mass spectrometric immunoassay (MSIA) MSIA is a unique

analytical technique which allows for the generation of distinct protein profiles of specific target proteins

from each subject which may be subsequently used in comparative protein expression profiling

between all subjects Comparative profiling allows for the rapid identification of variations within

individual protein expression profiles Although the majority of analyses performed in this studyrevealedhomology between study participants roughly one-quarter showed variation in the protein profiles

Some of these observed variants included a point mutation in TTR absence of wild-type RBP

monomeric forms UP1 a novel 2m glycated end product and altered HNP ratios MSIA has been

previously used in the analysis of blood proteins but this study shows how M SIA easily transitions to

the analysis of urine samples This study displays how qualitative urine protein differentiation is readily

achievable with MSIA and is useful in identifying proteomic differences between subjects that might

be otherwise overlooked with other analytical techniques due to complexity of the resulting data or

insufficient sensitivity

Keywords proteomics bull protein variations bull MALDI-TOF bull urine bull biomarker discovery

IntroductionUrine is an easily accessible biological fluid that has lately

become more intensely studied in the quest to identify protein

and peptide biomarkers that may potentially be used to assess

kidney function and identify the presence of disease in the

individual Many small proteins and peptides freely pass though

the glomerulus where they are then either catabolized within

the tubular cells of the kidney or are excreted in the urine1

Abnormalities in kidney function and the presence of disease

often result in variations in urine protein excretion rate and

content both of which have been historically monitored via

enzyme-linked immunosorbent assays (ELISA)23 This along

with the fact that the acquisition of urine is normally a

noninvasive procedure makes it an ideal biological fluid for

human proteomics studiesThe field of proteomics is developing new technologies and

methodologies toward the analysis of proteins from a variety

of biological fluids including urine A common proteomic

approach to analyzing urine proteins involves 2-dimensional

polyacrylamide gel electrophoresis (2D-PAGE) for protein

separation Even though this method is capable of separating

hundreds to thousands of proteins in a single analysis it is not

without weakness 2D-PAGE has in the past had poor results

in the analysis of peptides due to their high mobilities4 The

identification of separated proteins as well as the detection of

low-abundance proteins has also been historically problematic

A more recent innovation which incorporates 2D-PAGE with

mass spectrometry (2DEMS) provides more accurate results

but requires enzymatic digestion of the isolated proteins5

Although able to accurately identify genes from which pro-

teolytic fragments originate6 the 2DEMS approaches are not

readily able to yield information on the full-length protein and

oftentimes subtle details on the analyte (eg the presence of

point mutations and post-translational modifications) are

missed Because this information can be lost analysis of intact

proteins is becoming more widely accepted as a mean of proteomic analysis7

A proteomics technology that has great potential in the area

of intact urine protein analyses is the mass spectrometric

immunoassay (MSIA) MSIA combines the selectivity of an

immunoassay with the sensitivity resolution and mass ac-

curacy of matrix assisted laser desorptionionization time-of-

flight mass spectrometry (MALDI-TOF MS) A major advantage

of mass spectrometric detection over other conventional im-

munoassay schemes is the ability to discriminate between

protein variants in a single assay8 Because retrieved species

To whom correspondence should be addressed Tel (480) 804-1778Fax (480) 804-0778 E-mail rnelsonintrinsicbiocom

dagger Intrinsic Bioprobes IncDagger Arizona State University

101021pr025574c CCC $2500 983209 2003 American Chemical Society J ournal of Proteome Research 2003 2 191-197 191

Published on Web 01162003



are detected at precise molecular masses mass-shifted variants

of a protein (ie post-translational modifications or point

mutations) are readily detected in a single assay This approach

is contrary to conventional immunoassays where each protein

variant would require an individual monospecific assay which

obviously requires an a priori knowledge of the variants under

investigation (ie a monospecific assay must be constructed

for each variant after the variant is either discovered or

hypothesized) Conversely the MSIA approach is able to

discover as yet unidentified variants of proteins through the useof pan antibodies towards the protein of interest Thus MSIA

when applied to the routine screening of known protein

variants (ie wild-type) holds much potential in the discovery

and identification of variants resulting from post-translational

modifications splicing variations or point mutations

Moreover MSIA in the form of an affinity pipettor tip is

capable of analyzing very low abundance proteins via a

repetitive pipetting action The flowing action of the pipettor

tip concentrates and purifies the target protein prior to MALDI-

TOF MS analysis allowing for routine analyses of protein targets

in the picomolar and sub-picomolar range9 Once in the mass

spectrometer different forms of the same protein are readily

distinguishable by measurable alterations in molecular mass

thus allowing for protein isoform identificationThe general utility of MSIA is not that of generating global

gene product expression profiles as done by many other

proteomics technologies but is that of targeting a specific

protein and analyzing all endogenous forms of the intact target

protein from within a specific human biological fluid In this

manner the exact form(s) of a proteinsrather than those

presumed from a genomic database searchscan be determined

within an individual and slight changes in structure discerned

for (ultimately) correlation with disease Previously MSIA has

been demonstrated in the high throughput quantification and

characterization of various human plasma proteins10-12 but still

has been largely unexploited in the field of urine protein

analyses with the only reported application being the quan-

titation of -2-microglobulin ( 2m)13 and the comparativeprofiling of retinol binding protein (RBP)14 Described here is

the development of further urine-based MSIA assays targeting

transthyretin (TTR) cystatin C (CYSC) urine protein 1 (UP1)

albumin (ALB) transferrin (TRFE) and human neutrophil

defensins (HNP) and a brief study illustrating their use in

profiling these proteins between individuals

Experimental Section

Study Subjects Urine samples were collected from 5 unre-

lated male subjects ages ranging from 26 to 79 Four subjects

ages 26-68 were healthy study participants whereas one

individual age 79 was diagnosed with pancreatic cancer Urine

samples were obtained via protocols approved through IntrinsicBioprobes Incrsquos Internal Review Board (IRB) The individuals

had read and signed an Informed Consent form

Sample Preparation Urine samples 25 mL mid-stream

voids from five individuals were collected The urine was

collected directly into sterile urine collection cups that were

pretreated with 50 microL of the protease inhibitor cocktail consist-

ing of AEBSF (100 mM) aprotin (80 microM) bestatin (5 mM) E-64

(15 mM) leupeptin (2 mM) pepstatin A (1 mM) to prevent

any enzymatic breakdown or modification Samples were

collected and stored at -70degC until ready for analysis Samples

were thawed in a warm water bath (37 degC) just prior to analysis

Sample Analysis Each sample was combined 11 (vv) with

2M ammonium acetate (to adjust the pH to sim68-72) and

poured into an individual poly(vinyl chloride) solution basin

prior to analysis Each sample was individually addressed with

individual MSIA-Tips (Intrinsic Bioprobes Inc) derivatized with

anti- 2m anti-TTR anti-CYSC anti-UP1 anti-RBP anti-ALB

anti-TRFE or carboxylic acid surface (cation exchange for HNP

analysis) All eight analyses were run in parallel through each

sample with MSIA-Tips loaded onto an octapette

The manual incubation of each urine sample consisted of

300 cycles (150 microL of sample) through each MSIA-Tip After

incubation tips were thoroughly rinsed using HBS buffer (10

cycles 150 microL) doubly distilled water (5 cycles 150 microL) 20

acetoniltrile1M ammonium acetate wash (10 cycles 150 microL)

and finally with doubly distilled water (15 cycles 150 microL)

Retained species were eluted by drawing 4 microL of MALDI matrix

solution (saturated aqueous solution of sinapic acid (SA) in

33 (vv) acetonitrile 04 (vv) trifluoroacetic acid) into eachtip and depositing directly onto a 96-well formatted hydro-

phobichydrophilic contrasting MALDI-TOF target10 Because

of the larger sample volumes of urine and the number of

iterations used the time spent to run the assays were sim20-

minutessample MALDI-TOF mass spectrometry and data

analysis were performed on all samples as described previ-

ously10 Acquired mass spectra all had mass accuracy within

001 which was sufficient to correctly identify target proteins

Results and Discussion

The analysis of urine directly with MALDI-TOF MS is shown

in Figure 1 The spectrum was produced by diluting human

urine (Individual 1) by a factor of 20 in double distilled water

and serves as a control (point of reference) for the MSIA process A dilution of urine was required in order to reduce

the high salt content of the biological fluid which would

otherwise disrupt the MALDI process Only small peptides are

observed

-2-Microglobulin ( 2m) 2m is a low molecular weight

protein (11 729 Da) which was identified as a light chain of

the class I major histocompatability antigens15 Found in most

biological fluids elevated levels of 2m in blood and urine can

result from a number of ailments (ie AIDS16 rheumatoid

arthritis17 leukemia18) Glycation of 2m the covalent attach-

ment of a reducing sugar is commonly observed in individuals

Figure 1 Mass spectrum of urine diluted 120 Small peptides

were present whereas target proteins were not observed

research articles Kiernan et al

192 J ournal of Proteome Research bull Vol 2 No 2 2003



with one of a number of metabolic disorders (ie diabetes

mellitus uremia hypoglycemia etc) resulting in advancedglycation end products (AGEs)1920 Moreover 2m has also

been pathogenically associated in many amyloid disorders

including dialysis related amyloidosis (DRA)21

Analyses of the human urine samples with anti- 2m MSIA

are shown in Figure 2 with wild-type (wt-) 2m seen in the mass

spectra of all study participants Inter-individual results are all

very similar as shown in Figure 2 inlet except for Trace E in

which a glycated 2m variant is present in relatively high

abundance Glycated 2m is the result of excess dietary

reducing sugars present in the blood nonenzymatically attach-

ing to free amine groups of local proteins through the ldquoMaillard

reactionrdquo that can result in a number of possible Amidori

products2223 The observed glycated 2m in Figure 2-Trace E

has a measured mass increase of sim146 Da which correspondsto an imidazolone formation an Amidori product consisting

of a cyclic ring formed between a reduced sugar and an arginine

residue This 2m variant was originally observed by Niwa et

al in amyloid plaque deposits22 but was then postulated to

be artifacts of the overexposure of the insoluble 2m plaques

to normal levels of blood reducing sugars The data presented

here is the first report of imidazolone-modified 2m from a

human biological fluid and demonstrates that this unique form

of glycation can be found on native unplaqued 2m The

individual whose results were shown in Trace E was diagnosed

with pancreatic cancer and clinical studies have shown a

strong correlation between the development of the pancreatic

cancer and diabetes mellitus 2425 Hence the presence of AGEs

in this patient might be suggestive of the presence of a

metabolic disease such as diabetes mellitus but due to a lack

of symptoms the patient was not tested at the time of sample

collection

Transthyretin (TTR) TTR the second target is a thyroid

hormone carrier found in high levels in both serum and

cerebral spinal fluid Produced mainly in the liver TTR forms

a homotetramer2627 and is often complexed with other proteins

in the transport of various biologically active compounds

Structurally wt-TTR comprises 127 amino acids and has a MW

of 13 762 Over 80 point mutations have been cataloged for

TTR with all but 10 potentially leading to severe complica-

tions28 The majority of these mutation-related disorders are

caused by amyloid plaques depositing on various tissues

eventually leading to complex dysfunctions including carpal

tunnel syndrome drussen and familial amyloid poly-

neuropathy29-32 Multiple TTR post-translational modifications

have been previously detected in the plasma of healthy

individuals33 due mostly to TTR having a free cysteine residue

which commonly react with other sulfhydrils cysteines glu-

tathions etcFigure 3 shows the result of the urinary MSIA analyses of

TTR Both wt- and post-translationally modified (PTM) forms

of intact TTR are readily apparent in all five traces This in itself

is novel due to conflicting reports regarding proximal tubular

reabsorption and the presence of TTR in urine3433 The most

abundant PTM observed is the cysteinylated (cys) form ∆m )

+119 Da Expanded views of the singly charged TTR signals

shown in each corresponding Figure 3 inlet clearly show that

ratios between the PTM and the wild-type forms of TTR vary

between all individuals analyzed Moreover the TTR signal

shown in Figure 3-Trace B exhibits peak splitting which is

indicative of the presence of a heterozygous point mutation

The resolution of the linear TOF MS (m ∆m ) sim1000) is

sufficient to determine the mass shift of the variant to be sim+

30 Da This approximate mass shift is accurate enough to

decrease number of possible variants from 80 to sim7 The results

shown in Figure 3-Trace B would normally warrant further

protein characterization ie enzymatic digestion however

previous MSIA plasma-based studies involving the same indi-

vidual have already determined the point mutation to be a

Thr119Met substitution11

Cystatin C (CYSC) Cystatin C (CYSC) is an extracellular

cysteine protease inhibitor found in most biological fluids Even

though CYSC is freely filtered by the glomerulus urinary levels

of CYSC are a poor marker for glomerular filtration due to

Figure2 Mass spectrometric results of urinary 2m MSIA Signal

from wild-type 2m is consistent in all five traces however Trace

E also contains a high proportion of glycated 2m Glycation

resulted in a + 146 Da mass shift due to the covalent modification

by a deoxyhexose Figure 3 Comparison of urinary TTR MSIA Both the wild-type

and a post-translationally modified (cysteinylation ∆m ) +128

Da) forms are seen in all five traces While varied amounts of

cysteinylated TTR are seen present in each sample TraceB alsoshows the presence of a variant form of the TTR protein Peak

splitting sim∆m ) +30 is observed in the wild-type and the

cysteinylated forms of TTR because of two forms of the protein

being expressed a w ild-type and mutant that are eventually

excreted into urine

C om p a r a t i v e U r i n e P r o t ei n P h en o t y pi n g research articles

J ournal of Proteome Research bull Vol 2 No 2 2003 193



tubular reabsorption35 but have been used as a reliable

measure of proximal tubular reabsorption which has been

linked to renal failure36 Moreover hereditary cerebral hemor-

rhage with amyloidosis (HCHWA) an autosomal dominant

disorder prevalent in Icelandic Dutch and Finish populations

is the result of a CYSC Leu68f Gln variant37 This variant of

CYSC results in amyloid deposits of the walls of cerebral

arteries A number of carcinoma cell lines have also been

reported to secrete CYSC leading to investigations of its role

as a possible tumor marker CYSC also has several PTMassociated with it most notable is the hydroxylation of a Pro

residue at position 336 which results in a mass shift of the wt-

CYSC protein by sim+16 Da

The results of the anti-CYSC MSIA analyses are shown in

Figure 4 in which very similar protein profiles are observed

between all subjects The mass spectrometric analysis was able

to sufficiently resolve the wt- (13 344 Da) and the hydroxylated

form (13 360 Da) of CYSC m ∆m ) sim1000 Varied amounts of

hydroxylation are seen between each individual Multiple

N-terminally truncated forms of CYSC are also present most

notable are the S- (13 256 Da) and SSP- (13 072 Da) Hy-

droxylation still occurs in the S- variant (13 272 Da) but is

lost with the cleavage of the P- at position 3 Further truncated

forms of CYSC are observed in Traces D and E in which

SSPGKPPR- (12 536 Da) SSPGKPPRL- (12 423 Da) and SSPGK-

PPRLV - (12 324 Da) are also present The degradation of CYSC

has been reported from a significant portion of native urine

samples to date36 but this CYSC profiling clearly shows that

this catabolic process is conserved a N-terminal proteolytic

process

Urine Protein 1 UP1 also known as Clara cell protein CC10

or uteroglobin is a biomarker for a variety of pulmonary

ailments and urinary tract dysfunctions UP1 is a small protein

MW ) 7909 that is primarily secreted by Clara cells in the

bronchi alveolar lining in mammalian lung tissue is an anti-

inflammatory agent38 but to date its physiological role is stilllargely unclear The native state of UP1 is a covalently associ-

ated homodimer which results from the disulfide cross bridg-

ing between two UP1 monomers39 When damage occurs to

the respiratory tract plasma and urine UP1 levels increase due

to increased bronchioalveolar permeability and the overloading

of the tubular reabsorption process respectively4041 Moreover

increased UP1 concentration in urine alone basal levels 5-10

microgL41 is often an indication of proximal tubular dysfunction42

whereas decreased UP1 plasma levels have been found in

smokers43 asthmatics44 and schizophrenics45

Figure 5 shows the results of the qualitative urinary UP1

MSIA analysis Dimerized UP1 with MW ) 15 819 is present in

all five traces Although multiple charging of UP1 during the

MALDI process is unable to directly differentiate betweenpotential UP1 monomers and the +2 state of the UP1 dimer

conjugated monomers are readily identifiable Because wt-UP1

monomer would have exposed free cysteine groups some sort

of chemical modification through these reactive sulfhydryls

would be expected as seen in TTR Closer examination in the

Figure 5 inlets shows that both Traces A and B contain UP1

monomer with varied amounts of glutathion conjugate (∆m )

+305 Da) associated Although being virtually undetectable in

Traces C-E this is the first reported incidence of UP1

monomer being detected These results suggest that the

individualsrsquo results shown in Traces A and B have more

glutathion andor UP1 monomer present in their systems

Whether this observation is associated with health state or

significant to disease has yet to be determined

Retinol Binding Protein (RBP) RBP was the fifth urine

protein target A member of the lipocalin family RBP has a

plasma concentration level of sim50 mgL and serves the role of

the major carrier of retinol (vitamin A) from the liver to

peripheral tissues46 With a molecular weight of 21 065 RBP is

believed to escape glomerular filtration by associating with the

homotetramer of transthyretin in its holo- (retinol bound)

form47 RBP has been previously reported to exist in two post-

translationally truncated versions one missing the C-terminal

Leucine (RBP-(Leu)) and the second missing two C-terminal

leucines (RBP-(Leu-Leu)) which are believed to be nonfunc-

Figure 4 Results of urinary CYSC MSIA analysis Both the wild-

type and hydroxylated (∆m ) +16Da) forms of CYSC are present

in all five traces Varied amounts of hydroxylated CYSC are seen

in each individual as well as multiple truncated forms of theprotein These truncations include the systematic N-terminal

cleavage of S- SSP- and SSPG- Extensive cleavage of CYSC

with the loss of SSPGKPPR- SSPGKPPRL- and SSPGKPPRLV-

are only seen in Traces D and E

Figure 5 Mass spectrometric results of urinary UP1 MSIA

Dimerized UP1 (MW ) 15 819) is seen in all five traces but Traces

A and B both contain large proportions of UP1 monomer

conjugated to glutathion (∆m ) +305 Da)





tional variants of RBP due to their lower binding affinities to

the transthyretin complex48

The results of the urinary RBP analysis are shown in Figure

6 Conserved protein profiles are seen in Traces A -D with wt-

RBP along with -L -LL -RNLL and -RSERNLL C-terminally

truncated variants The source and function of these variants

are still unknown but have been determined to be the result

of some unreported enzymatic process that occurs after the

RBP is filtered from the blood14 Interestingly the individual

with pancreatic cancer in Figure 6-Trace E displays an altered

RBP profile Most notable is the marked relative decrease in

the amount of wt-RBP present Similar results were reported

with the analysis of urinary RBP of a 94-year old woman with

renal failure stemming from chronic diabetes mellitus in which

wt-RBP was completely absent from the RBP protein expression

profile14

Albumin (ALB) ALB the sixth urine protein target is the

best studied of all plasma proteins49 At sim663 kDa ALB is

considerably larger than any of the previously discussed protein

targets As a multipurpose house-keeping protein ALB serves

a multitude of functions including the binding and transport

of many metallic organic and biochemical compounds anti-

oxidant effects as well as plasma buffering5051

Figure 7 shows the results of the urinary ALB MSIA with the

+1 and the +2 states of ALB are present in all traces Inter-

individual results are all very similar as shown in Figure 7 inlet

except for Trace E in which extensive peak broadening is

observed Since albumin participates in the transport of so

many biological inorganic and pharmacological compounds

adduct formation with one or many of these compounds is

possible Albumin is also known to undergo glycation like 2m

hence the observed peak broadening may be the result of the

formation of an ALB-AGErsquos

Transferrin (TRFE) TRFE the seventh protein target is a

large globular glycoprotein (MW ) 796 kDa) used in the

transport of dietary iron in human plasma52 TRFE readily

crosses the glomerular membrane despite its large size due

to its strong cationic nature53 resulting in urine levels lt019

mgL54 With two N-linked glycosylation sites the heterogeneity

within the sim44 kDa of associated glycan can greatly vary

Figure 8 displays the results of the anti-TRFE MSIA analysis

Strong homology is seen in the results in all five Traces This

homology in TRFE shows that chronic alcoholizm and carbo-

hydrate deficient glycoprotein syndromes (CDGSI or II) were

not present in any of the participants of this study Only Traces

B and E have detectable amounts of TRFE in the +2 state

whereas Trace A does exhibit some tailing in the TRFE signal

Whether these minute differences seen in the TRFE profiles

are related to any disease state or from genetic modificationhas yet to be determined

Human Neutrophil Defensin Peptides (HNP) HNP also

known as the R -defensins was the final urine target in this

study HNP are a family of cysteine-rich cationic peptides 29-

42 amino acids in length secreted from neutrophils The three

most common human neutrophil defensin peptides are HNP-1

(MW ) 3443) HNP-2 (MW ) 3372) and HNP-3 (MW ) 3487)

which are found in plasma urine saliva and sputum5556 These

peptides have demonstrated remarkable antibacterial antifun-

gal and antiviral activities thus suggesting that HNP plays as

strong role innate immunity55

Figure 6 MSIA results of RBP analysis Conserved C-terminal

cleavage pattern is seen in TracesA-D with theloss of -L -LL

-RNLL and -RSERNLL Trace E displays an abnormal RBP

profile due to the noticeable decrease in the amount of wt-RBP

present compared to the truncated forms

Figure 7 Comparison of ALB MSIA analysis ALB +1 and +2

states were detected in all five samples Even though all traces

were acquired using the same instrument settings Trace E

exhibits significant peak broadening of the ALB signal shown

in trace insets as compared to the other ALB signals in the other

traces

Figure 8 Results of TRFE MSIA analysis TRFE (MW ) 796kDa)

was detected in all five samples Only very minor differences

were observed





Previous studies into the roles of HNP in various systemic

responses have shown that they partake in large number of

responses including pro-inflammation with histamine release57

as well as cell proliferation and mitogenic effects58 Because of

this last effect more recent studies have suggested a correlation

between HNP and certain cancers59 Other studies have shown

that HNP may also serve as a marker for certain inflammatory

disease states and sepsis6061

Because these peptides are highly cationic immuno-affinity

retrieval is not required Cation exchange MSIA ionically retains

sufficient amounts of HNP to allow for mass spectrometric

detection The results of this analysis are shown in Figure 9 in

which all three major HNP species were detected in all five

samples as shown by the triple peaks Intra-sample concentra-

tion of each HNP species varies from each sample Traces C

and E show that these individuals had more HNP-2 relative to

the other two HNP forms than the other individuals involved

in this study Whether the HNP species ratios are linked to

disease states or are subject to diurnal variation has yet to be

determined

The results of all 40 analyses were summarized for compari-

son in Table 1 which describes structural variations observed

in the target proteins Interestingly the individual diagnosed

with pancreatic cancer showed the most protein variation

compared to the other samples The presence of glycated 2m

and the decrease in wt-RBP were most prominent of all the

differences Similar types of variations have been previously

described from individuals with diabetes mellitus andor ure-

mia but neither of these conditions had been previously

diagnosed in this individual Moreover broadening of this

individualrsquos ALB signal was also observed and may be associ-

ated to albuminrsquos function in bio- and organic-molecule

transport whereas other differences included the increase

truncation of CYSC and altered ratios of HNP-1 and -2 were

seen but not isolated to this individual The analysis of TTR

showed that one individual had a heterozygous point mutation

demonstrating the utility of MSIA in the identification of genetic

variation at the protein level The application of MSIA was ableto also identify monomeric forms of UP1 modified with

glutathion in two individuals In total approximately one-

quarter of the analyses provided variant results from the wild-

type

Conclusion

This study demonstrates that mass spectrometric immu-

noassay is a powerful analytical technique in the study of intact

urinary proteins MSIA allows for the rapid retrieval of specific

protein targets which permits concise identification of each

target species to be achieved Unlike indirect detection as used

in ELISAs mass spectrometry is able to discriminate between

variant forms of a protein target that are present making

identification of all species possible Moreover MSIA is an

analytical technique that allows for comparative protein profil-

ing to look for differences between individuals in specific

protein targets Many of these differences are subtle and could

not be readily distinguished without mass spectrometric detec-

tion As shown here a small study with 40 data points produced

almost a dozen observable differences between 5 individuals

The analysis of 8 protein targets detected 29 different observed

forms of these proteins making mass spectrometry integral for

protein phenotyping The observation of such variation within

such a small study population necessitates the need for larger

urine protein population studies in order to correlate such

findings to possible disease states

Acknowledgment This publication was supported in

part by Grant No R44 GM56603-01 and Contract No N43-DK-

1-2470 from the National Institutes of Health Its contents are

solely the responsibility of the authors and do not necessarily

represent the official views of the National Institute of Health

References

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(2) Tomlinson P A Dalton R N Turner C Chantler C ClinChim Acta 1990 192 99-106

Table 1 Summary of Protein Profile of All Eight Assays Run on All Five-Study Subjectsa

2m TTR CYTC UP1 RBP ALB TRFE HNP

sample A o o o glutathionconjugated

monomer

o o tailing o

sample B o pointmutation

o glutathionconjugated

monomer

o o o o

sample C o o o o o o o altered

ratiossample D o o extended

truncations

o o o o o

sample E glycation o extendedtruncations

o decreased wt-RBP

peak broadening

o alteredratios

Figure 9 Mass spectrometric results of HNP analysis Signals

from HNP-1 (MW ) 3443) -2(MW ) 3372) and -3(MW ) 3487)

were detected in all samples Differences in relative amounts of

HNP-1 to HNP-2 are observed between each sample





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P Sleeman J Lamond A Mann M Nat Genet 1998 20 46-

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1995 67 1153-1158

(9) Kiernan U A Doctoral Dissertation Arizona State University2002

(10) Niederkofler E E Tubbs K A Gruber K Nedelkov D KiernanU A Williams P Nelson R W Anal Chem 2001 73 3294-

3299(11) Kiernan U A Tubbs K A Gruber K Nedelkov D Niederkof-

ler E E Williams P Nelson R W Anal Biochem 2002 30149-56

(12) Kiernan U A Nedelkov D Tubbs K A Niederkofler E ENelson R W Am Biotech Lab 2002 20 26-28

(13) Tubbs K A Nedelkov D Nelson R W Anal Biochem 2001289 26-35

(14) Kiernan U A Tubbs K A Nedelkov D Niederkofler E ENelson R W Biochem Biophys Res Commun 2002 297 401

(15) Schardijn G H Statius van Eps L W Kidney Int 1987 32 635-

41(16) Lifson A R Hessol N A Buchbinder S P OrsquoMalley P M

Barnhart L Segal M Katz M H Holmberg S D Lancet 1992

339 1436-40(17) Walters M T Stevenson F K Goswami R Smith J L Cawley

M I Ann Rheum Dis 1989 48 905-11(18) Sadamori N Mine M Hakariya S Ichiba M Kawachi T

Itoyama T Nakamura H Tomonaga M Hayashi K Leukemia 1995 9 594-7

(19) Schwedler S B Metzger T Schinzel R Wanner C Kidney Int2002 62 301-310

(20) Vlassara H Palace M R J Intern Med 2002 251 87-101(21) Kleinman K S Coburn J W Kidney Int 1989 35 567-75(22) Niwa T Katsuzaki T Miyazaki S Momoi T Akiba T

Miyazaki T Nokura K Hayase F Tatemichi N Takei YKidney Int 1997 51 187-94

(23) Humeny A Kislinger T Becker C M Pischetsrieder M J AgricFood Chem 2002 50 2153-2160

(24) Ogawa Y Tanaka M Inoue K Yamaguchi K Chijiiwa KMizumoto K Tsutsu N Nakamura Y Cancer 2002 94 2344-

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(25) Wakasugi H Funakoshi A Iguchi H Int J Clin Oncol 20016 50-4

(26) Ingenbleek Y Young V Annu Rev Nutr 1994 14 495-533(27) Schreiber G Richardson S J Comp Biochem Physiol B

Biochem Mol Biol 1997 116 137-60(28) Connors L H Richardson A M Theberge R Costello C E

Amyloid 2000 7 54-69(29) Damas A M Saraiva M J J Struct Biol 2000 130 290-9(30) Mullins R F Russell S R Anderson D H Hageman G S

Faseb J 2000 14 835-46(31) Plante-Bordeneuve V Said G Curr Opin Neurol 2000 13 569-

73(32) Benson M D Uemichi T Amyloid-Int J Exp Clin Invest 1996

3 44-56(33) Terazaki H Ando Y Suhr O Ohlsson P I Obayashi K

Yamashita T Yoshimatsu S Suga M Uchino M Ando MBiochem Biophys Res Commun 1998 249 26-30

(34) Jacobsson B Lignelid H Bergerheim U S Histopathology 199526 559-64

(35) Woitas R P Stoffel-Wagner B Poege U Schiedermaier PSpengler U Sauerbruch T Clin Chem 2001 47 2179-80

(36) Grubb A O Adv Clin Chem 2000 35 63-99(37) Olafsson I Grubb A Amyloid 2000 7 70-9(38) Dierynck I Bernard A Roels H De Ley M Mult Scler 1996

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PR025574C





are detected at precise molecular masses mass-shifted variants

of a protein (ie post-translational modifications or point

mutations) are readily detected in a single assay This approach

is contrary to conventional immunoassays where each protein

variant would require an individual monospecific assay which

obviously requires an a priori knowledge of the variants under

investigation (ie a monospecific assay must be constructed

for each variant after the variant is either discovered or

hypothesized) Conversely the MSIA approach is able to

discover as yet unidentified variants of proteins through the useof pan antibodies towards the protein of interest Thus MSIA

when applied to the routine screening of known protein

variants (ie wild-type) holds much potential in the discovery

and identification of variants resulting from post-translational

modifications splicing variations or point mutations

Moreover MSIA in the form of an affinity pipettor tip is

capable of analyzing very low abundance proteins via a

repetitive pipetting action The flowing action of the pipettor

tip concentrates and purifies the target protein prior to MALDI-

TOF MS analysis allowing for routine analyses of protein targets

in the picomolar and sub-picomolar range9 Once in the mass

spectrometer different forms of the same protein are readily

distinguishable by measurable alterations in molecular mass

thus allowing for protein isoform identificationThe general utility of MSIA is not that of generating global

gene product expression profiles as done by many other

proteomics technologies but is that of targeting a specific

protein and analyzing all endogenous forms of the intact target

protein from within a specific human biological fluid In this

manner the exact form(s) of a proteinsrather than those

presumed from a genomic database searchscan be determined

within an individual and slight changes in structure discerned

for (ultimately) correlation with disease Previously MSIA has

been demonstrated in the high throughput quantification and

characterization of various human plasma proteins10-12 but still

has been largely unexploited in the field of urine protein

analyses with the only reported application being the quan-

titation of -2-microglobulin ( 2m)13 and the comparativeprofiling of retinol binding protein (RBP)14 Described here is

the development of further urine-based MSIA assays targeting

transthyretin (TTR) cystatin C (CYSC) urine protein 1 (UP1)

albumin (ALB) transferrin (TRFE) and human neutrophil

defensins (HNP) and a brief study illustrating their use in

profiling these proteins between individuals

Experimental Section

Study Subjects Urine samples were collected from 5 unre-

lated male subjects ages ranging from 26 to 79 Four subjects

ages 26-68 were healthy study participants whereas one

individual age 79 was diagnosed with pancreatic cancer Urine

samples were obtained via protocols approved through IntrinsicBioprobes Incrsquos Internal Review Board (IRB) The individuals

had read and signed an Informed Consent form

Sample Preparation Urine samples 25 mL mid-stream

voids from five individuals were collected The urine was

collected directly into sterile urine collection cups that were

pretreated with 50 microL of the protease inhibitor cocktail consist-

ing of AEBSF (100 mM) aprotin (80 microM) bestatin (5 mM) E-64

(15 mM) leupeptin (2 mM) pepstatin A (1 mM) to prevent

any enzymatic breakdown or modification Samples were

collected and stored at -70degC until ready for analysis Samples

were thawed in a warm water bath (37 degC) just prior to analysis

Sample Analysis Each sample was combined 11 (vv) with

2M ammonium acetate (to adjust the pH to sim68-72) and

poured into an individual poly(vinyl chloride) solution basin

prior to analysis Each sample was individually addressed with

individual MSIA-Tips (Intrinsic Bioprobes Inc) derivatized with

anti- 2m anti-TTR anti-CYSC anti-UP1 anti-RBP anti-ALB

anti-TRFE or carboxylic acid surface (cation exchange for HNP

analysis) All eight analyses were run in parallel through each

sample with MSIA-Tips loaded onto an octapette

The manual incubation of each urine sample consisted of

300 cycles (150 microL of sample) through each MSIA-Tip After

incubation tips were thoroughly rinsed using HBS buffer (10

cycles 150 microL) doubly distilled water (5 cycles 150 microL) 20

acetoniltrile1M ammonium acetate wash (10 cycles 150 microL)

and finally with doubly distilled water (15 cycles 150 microL)

Retained species were eluted by drawing 4 microL of MALDI matrix

solution (saturated aqueous solution of sinapic acid (SA) in

33 (vv) acetonitrile 04 (vv) trifluoroacetic acid) into eachtip and depositing directly onto a 96-well formatted hydro-

phobichydrophilic contrasting MALDI-TOF target10 Because

of the larger sample volumes of urine and the number of

iterations used the time spent to run the assays were sim20-

minutessample MALDI-TOF mass spectrometry and data

analysis were performed on all samples as described previ-

ously10 Acquired mass spectra all had mass accuracy within

001 which was sufficient to correctly identify target proteins

Results and Discussion

The analysis of urine directly with MALDI-TOF MS is shown

in Figure 1 The spectrum was produced by diluting human

urine (Individual 1) by a factor of 20 in double distilled water

and serves as a control (point of reference) for the MSIA process A dilution of urine was required in order to reduce

the high salt content of the biological fluid which would

otherwise disrupt the MALDI process Only small peptides are

observed

-2-Microglobulin ( 2m) 2m is a low molecular weight

protein (11 729 Da) which was identified as a light chain of

the class I major histocompatability antigens15 Found in most

biological fluids elevated levels of 2m in blood and urine can

result from a number of ailments (ie AIDS16 rheumatoid

arthritis17 leukemia18) Glycation of 2m the covalent attach-

ment of a reducing sugar is commonly observed in individuals

Figure 1 Mass spectrum of urine diluted 120 Small peptides

were present whereas target proteins were not observed



































collection





















































excreted into urine

































process










































































profile14






















































traces



were observed
























determined





















type

Conclusion


























References






monomer

o o tailing o



monomer

o o o o



truncations

o o o o o


o decreased wt-RBP

peak broadening

o alteredratios















1995 67 1153-1158



















2349




































PR025574C



































collection





















































excreted into urine

































process










































































profile14






















































traces



were observed
























determined





















type

Conclusion


























References






monomer

o o tailing o



monomer

o o o o



truncations

o o o o o


o decreased wt-RBP

peak broadening

o alteredratios















1995 67 1153-1158



















2349




































PR025574C

































process










































































profile14






















































traces



were observed
























determined





















type

Conclusion


























References






monomer

o o tailing o



monomer

o o o o



truncations

o o o o o


o decreased wt-RBP

peak broadening

o alteredratios















1995 67 1153-1158



















2349




































PR025574C




















profile14






















































traces



were observed
























determined





















type

Conclusion


























References






monomer

o o tailing o



monomer

o o o o



truncations

o o o o o


o decreased wt-RBP

peak broadening

o alteredratios















1995 67 1153-1158



















2349




































PR025574C
























determined





















type

Conclusion


























References






monomer

o o tailing o



monomer

o o o o



truncations

o o o o o


o decreased wt-RBP

peak broadening

o alteredratios















1995 67 1153-1158



















2349




































PR025574C











1995 67 1153-1158



















2349




































PR025574C



kiernan et al_2002_journal of proteome research_comparative urine phenotyping.pdf

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