Analytical Biochemistry 301, 49 56 (2002)

doi:10.1006/abio.2001.5478, available online at http://www.idealibrary.com on

High-Throughput Protein Characterization Using Mass

Spectrometric Immunoassay
Urban A. Kiernan,* , Kemmons A. Tubbs,* Karl Gruber,* Dobrin Nedelkov,*
Eric E. Niederkofler,* , Peter Williams, and Randall W. Nelson* ,1
*Intrinsic Bioprobes, Inc., 625 S. Smith Road Suite, 22, Tempe, Arizona 85281; and Department of Chemistry
and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604

Received May 29, 2001; published online December 10, 2001

A high-throughput mass spectrometric immunoassay system for the analysis of proteins directly from
plasma is reported. A 96-well format robotic workstation was used to prepare antibody-derivatized affinity
pipette tips for subsequent use in the extraction of
specific proteins from plasma and deposition onto 96well format matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF
MS) targets. Samples from multiple individuals were
screened with regard to the plasma protein transthyretin (TTR), followed by analysis of the same plasma
samples for the transthyretin-associated transport
protein, retinol-binding protein (RBP). Analyses were
able to detect the presence of posttranslationally modified TTR and RBP, as well as a mutation present in
the TTR of one individual. Subsequent analyses of
wild-type and mutated TTR using enzymatically active MALDI-TOF MS targets were able to identify the
site and nature of the point mutation. The approach
represents a rapid (100 samples/2 h, reagent preparation-to-data) and accurate means of characterizing
specific proteins present in large numbers of individuals for proteomic and clinical/diagnostic purposes.
2001 Elsevier Science

Key Words: MALDI-TOF; mass spectrometry; highthroughput; affinity; immunoassay; protein.

Present day proteomics relies heavily on the mass

spectrometric identification of proteins separated using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) 2 or tandem high performance liquid
1
To whom correspondence should be addressed. Fax: (480) 8040778. E-mail: rnelson@intrinsicbio.com.
2
Abbreviations used: 2D-PAGE, two-dimensional polyacrylamide
gel electrophoresis; MALDI-TOF MS, matrix-assisted laser desorp-

0003-2697/01 $35.00
2001 Elsevier Science
All rights reserved.

chromatography (LC/LC) approaches (1 4). Such analyses are impressive in their ability to simultaneously
interrogate hundreds to thousands of proteins present
in complex biological media. When used in control versus disease-state studies, the approaches are able to
identify proteins participating in, or serving as markers for, disease states. The identification process serves
as the starting point for subsequent studies geared
toward, among other things, the more detailed structural characterization of proteins. Due to the numerous genetic, transcriptional, and posttranslational
variations possible for any given protein, it is essential
that such follow-up studies investigate large numbers
of samples and are able to characterize the structure of
a protein to the fullest degree possible.
Once a protein is identified and deemed relevant to
further study, it is possible to replace 2D PAGE and
LC/LC with affinity selection in preparation for direct
mass spectrometric characterization. In this manner,
proteins of interest are extracted from the biological
medium to the exclusion of the rest of the complex
mixture. Ideally, the extraction process is free of hindrances from nonspecific binding of other proteins/compounds and exhibits an overall concentrating effect
toward the targeted protein. Indeed, numerous past
reports have demonstrated the potential of using affinity isolation in combination with mass spectrometry for
the analysis of proteins (518). Many of these reports
focus on the use of matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry (MALDITOF MS), which is used universally in the identification of affinity-isolated proteins (11, 12), the

tion/ionization time-of-flight mass spectrometry; MSIA, mass spectrometric immunoassay; b2m, -2-microglobulin; TTR, transthyretin; RBP, retinol binding protein; GA, glutaraldehyde; TFA,
trifluoroacetic acid; wt, wild type; MW, molecular weight.
49

50

KIERNAN ET AL.

investigation of posttranslational modifications (13),

the differential display of proteins (14), and the quantification of proteins retrieved directly from biological
media (8, 1518).
Because of its directness in sample preparation, affinity-isolation mass spectrometry represents an attractive means for second phase proteomics, where
the same protein is repetitively under investigation.
Such analyses have great potential in validation or
protein phenotyping studies where large populations
are under investigation and in more far-reaching application such as diagnostics. However, in order to
make full use of the speed of the approach, it is necessary to combine the affinity isolation/sample preparation with robotics. In this manner, high-throughput
platforms (e.g., 96-well format titer plates) can be used
to address multiple samples in parallel. Such use of
robotics in parallel processing requires the development of specialized affinity isolation devices and associated methods tailored specifically for the MALDITOF MS analysis. Moreover, to reach an additional
level of analytical potential, it is often necessary to
enzymatically digest small amounts of isolated proteins in order to gain a more detailed mass spectrometric characterization. Such processing requires yet
another set of specialized methods and devices for
high-throughput application.
Recently, we have described prototype affinity pipette tips used in the mass spectrometric immunoassay (MSIA) (17) of -2-microglobulin (b2m) present in
various human biofluids (15). The affinity pipette tips
were found to perform exceptionally well in the quantification of b2m present in urine and overall represented a vast improvement over rudimentary affinity
pipette tips described previously (18). Additional improvements upon the prototype design have yielded
affinity pipette tips that performed equally well in
other biofluids, in particular plasma (16). Parallel to
the development of affinity pipette tips has been the
development of enzymatically active MALDI-TOF targets for use in characterizing proteins postisolation
(19 21). These activated targets serve as enzymatic
modifiers of proteins, as well as platforms for introduction of samples into the mass spectrometer. Formatted
to 96-well platforms, both the affinity pipette tips and
the enzymatically active targets serve as universal devices that can be coupled to robotics for the processing
of numerous samples in parallel (see Fig. 1).
Presented here are high-throughput protein phenotyping studies investigating the use of a parallel robotic system equipped with affinity pipette tips for
preparing transthyretin (TTR) and retinol binding protein (RBP) directly from human plasma for MALDITOF MS characterization. Moreover, when needed, enzymatically active targets were included into the
analyses for a more detailed structural analysis of the

FIG. 1. High-throughput robotics platform for automated sample

preparation and MSIA analysis. Analytical samples are addressed in
parallel using a 96-well robotic workstation fitted with affinity pipette tips (1). A six-stage work area for automated processing enables sample incubation through the tips (repetitive aspiration and
dispensing, stage 2), rinsing (stages 3 and 4), and MALDI matrix
aspiration (stage 5). The final step of the high-throughput MSIA is
the elution of the matrix/analyte mix from the affinity pipette tips
directly onto a 96-well format hydrophobic/hydrophilic contrasting
MALDI-TOF MS target (stage 6).

targeted proteins. Of particular interest in the study

were: (1) the construction of affinity pipette tips exhibiting low nonspecific binding properties when applied
to plasma proteins, (2) the development of high-sensitivity MSIA protocols that allowed minimally invasive
sampling protocols, and (3) the identification of posttranslational modifications and point mutations
present in TTR and RBP.
MATERIALS AND METHODS

Affinity Pipette Tip Preparation

Affinity pipette tips were prepared following one of
three methods. All three methods utilized porous
amine frits described previously (15). In brief, the frits
were produced in bulk by loading soda lime glass beads
into stainless steel annealing molds and baked to form
a solid, yet porous frit. The frits were then removed
and acid conditioned prior to a 12-h treatment with
10% aminopropyl triethoxysilane. Once treated, the
frits are left with a functional amine surface that can
be used for coupling of ligands. In the first method,
amine frits were activated by 19-h exposure to glutaraldehyde (GA) (25% solution in 0.2 M phosphate buffer,
pH 7.5) followed by rinsing (3 50 mL) with phosphate
buffer. The activated frits were then incubated with
rabbit anti-TTR polyclonal antibody (Dako, Carpinteria, CA; Catalog No. A0002), diluted fivefold in HBS
buffer (0.01 Hepes, pH 7.4, 0.15 M NaCl, 0.005% (v/v)
polysorbate 20, 3 mM EDTA), for 19 h at 4C. Follow-

HIGH-THROUGHPUT PROTEIN ANALYSIS

ing the antibody linkage, the frits were rinsed vigorously with HBS and packed while still wet into widebore P-200 pipette tips and stored in HBS buffer. In the
second method, frits were activated with GA as in the
first method and incubated with 40-kDa polylysine (5.0
mg/mL in phosphate buffer) for 19 h with gentle agitation. The frits were rinsed vigorously with phosphate
buffer and then activated with GA, derivatized with
antibody, and packed into pipette tips using the procedures described for the first method. Using the third
method, amine frits were packed into the pipette tip for
subsequent in-robotic (Beckman, Multimek 96, Fullerton, CA) amplification and antibody linkage. The tips
were first amplified by repetitively flowing GA (25%
solution in HBS, pH 7.5) through the tips (100 L, 120
repetitions), followed by rinsing with HBS (100 L, 20
repetitions), exposure to polylysine (0.1 mg/mL in HBS,
100 L, 120 repetitions), and rinsing (HBS, 100 L, 20
repetitions). The amplified tips were then activated
with GA (25% solution in HBS, pH 7.5, 100 L, 120
repetitions), exposed to anti-TTR antibody (0.05
mg/mL in HBS, 20 L, 120 repetitions), and rinsed
with HBS (100 L, 20 repetitions). The same in-robotic
procedure was used for the preparation of anti-RBP
tips using rabbit anti-RPB polyclonal IgG (Dako, Catalog No. A0054). The total time needed for in-robotic
derivatization was approximately 50 min.
Sample Preparation
Whole blood samples were collected from six healthy
male individuals. Samples (44.7 L) were acquired
under sterile conditions through a lancet-punctured
finger using a heparinized microcolumn (Drummond
Scientific Co., Broomall, PA). The whole blood was
immediately combined with 200 L HBS and centrifuged for 1 min (at 7000 RPM, 2500g) to pellet red
blood cells. The supernatant (plasma/HBS) from each
sample was distributed in 25-L aliquots randomly
among the 96 wells of a titer plate, each well already
containing 100 L of HBS.
The plasma/HBS samples were addressed in parallel
using the 96-well format pipetting workstation
equipped with either anti-TTR or anti-RBP affinity
pipette tips. Sample incubation consisted of 20 cycles
(aspiration and dispensing) of 100 L of each sample
through the affinity pipette tips. After incubation, tips
were rinsed using HBS (5 cycles, 100 L), acetonitrile
(3 cycles, 100 L), and doubly distilled water (10 cycles,
100 L). Retained species were eluted by drawing 4 L
of MALDI matrix solution (saturated aqueous solution
of -cyano-4-hydroxycinnamic acid, in 33% (v/v) acetonitrile, 0.2% (v/v) trifluoroacetic acid) into the tips and
depositing it directly onto a 96-well formatted MALDITOF target that had been treated as previously described (16). Samples were allowed to air dry prior to

51

target insertion into the mass spectrometer. The total

time required for preparation of the 96 samples was
approximately 10 min.
TTR Mapping
TTR was isolated from plasma samples using the
protocols listed above. TTR was eluted from the tips
using an acid solution (0.2% TFA, containing 1 mM
n-octylglucoside) in place of the MALDI matrix solution and applied directly to the trypsin-activated
MALDI-TOF MS target prepared as previously described (2123). Digests were allowed to proceed for 15
min at 40C in high humidity enclave before termination by the addition of 2 L of matrix solution. Samples
were air dried prior to insertion of the MALDI-TOF
target into the mass spectrometer.
Mass Spectrometry
MS analysis was performed on a Bruker Biflex III
MALDI-TOF mass spectrometer operating in linear
delayed-extraction mode with 19.00 kV full accelerating potential. Draw-out pulses of 1.700 (300-ns delay)
and 1.600 kV (300-ns delay) were used for TTR (singly
charged) and RBP (doubly charged), respectively. Mass
spectra were acquired automatically by summing ten
10 laser shot spectra while gauging spectra quality
using fuzzy logic routines (24). Mass maps (100 laser
shots) were acquired in reflectron mode using an instrument setting of full accelerating potential of 19.00
kV, an ion-mirror voltage of 20.00 kV, and a draw-out
pulse voltage of 1.65 kV (300-ns delay).
RESULTS AND DISCUSSION

Transthyretin is a small protein produced in the

liver and found in serum and cerebral spinal fluid as a
homotetramer (25, 26). Functionally, TTR serves unaccompanied in the transport of thyroid hormones or in
complexes with other proteins in the transport of various biologically active compounds. Structurally, wildtype (wt) TTR is composed of 127 amino acids and has
a molecular weight (MW) of 13,762.4. Over 80 point
mutations have been cataloged for TTR, with all but 10
potentially leading to severe neurological complications (26). The majority of mutation-related disorders
are caused by amyloid plaques depositing on neurons
or tissue, eventually leading to dysfunctions including
carpal tunnel syndrome and familial amyloid polyneuropathy (2729). Retinol binding protein is composed of
182 amino acids (MW (wt) 21,065.6) and is produced
primarily in the liver (30, 31). RBP, in a 1:1 complex
with TTR (one RBP molecule bound to one TTR tetramer) (32, 33), is a specific carrier of retinol (vitamin
A) from the liver stores to the peripheral tissues (34).
Several variants/mutations have been found to disrupt

52

KIERNAN ET AL.

FIG. 2. Affinity pipettes optimization. (A) Mass spectrum of diluted human plasma. (B) Mass spectrum obtained using an affinity pipette
tip prepared via bulk derivatization by glutaraldehyde (GA) activation of amine frits, followed by exposure of the frits to anti-TTR antibody
and loading of the affinity frits into pipette tips. (C) Mass spectrum obtained using an affinity pipette tip prepared via bulk derivatization
by GA activation of amine frits and linkage of 40-kDa polylysine, followed by GA activation of the polylysine, linkage of anti-TTR antibody
and loading of the affinity frits into the pipette tips. (D) Mass spectrum obtained using an affinity pipette tip prepared via robotic
derivatization by GA activation of amine frits and linkage of 40-kDa polylysine, followed by GA activation of the polylysine and linkage of
anti-TTR antibody. (E) Mass spectrum obtained from an affinity pipette tip prepared in the same manner as in (D), except for the use of
anti-RBP antibody.

the TTR:RBP complex, which in turn leads to diminished transport of vitamin A. These variants include
point mutations in both TTR (Ile84Ser) (35) and RBP
(Ile41Asn and Gly74Asp in different alleles in a single
individual) (36) and posttranslationally truncated versions of RPB (two variants: one missing the C-terminal
leucine (RBP-C-Leu) and the second missing two Cterminal leucines (RBP-C-LeuLeu) (37).
Affinity Pipette Tip Optimization
Initial studies were performed to optimize the affinity pipette tips for the direct extraction of TTR and
RBP from plasma. Based on past findings (16), aminefunctionilized pipette tips were chosen as base chemistries from which additional derivatization schemes
were applied and tested. Figure 2 shows a comparison
of methods for preparing the anti-TTR affinity pipette
tips. Figure 2A is a mass spectrum of diluted human
plasma, which is given to illustrate the need to extract
TTR and RBP from plasma in preparation for MALDITOF MS characterization. Signals from neither TTR
nor RBP are observed in the spectrum. Figure 2B is a
spectrum obtained using an affinity pipette tip prepared by glutaraldehyde activation of amine frits, followed by exposure of the frits to anti-TTR antibody.

Both the activation and the antibody linkage steps

were performed in bulk prior to loading of the affinity
frits into pipette tips. TTR signals are observed in the
spectrum; however, in the presence of other signals
attributed to the nonspecific binding of apolipoproteins
C-I, C-I and C-III. Figure 2C shows a spectrum obtained using an affinity pipette tip prepared by GA
activation of amine frits and linkage of 40-kDa polylysine, followed by GA activation of the polylysine and
linkage of anti-TTR antibody. All activation and coupling steps were performed in bulk prior to loading of
the affinity frits into the pipette tips. The spectrum
demonstrates a reduction of nonspecific binding and a
high-quality signal profile for the TTR. Figure 2D
shows a spectrum obtained from an affinity pipette tip
to which polylysine-intermediate and antibody were
coupled using a robotic protocol. In this method, amine
frits were prepared in bulk and loaded into pipette tips
and the fully assembled tips were then loaded into a
96-well format robotic workstation. Polylysine intermediate was coupled to the tips (via GA activation)
followed by antibody linkage (through GA activation
and exposure to anti-TTR antibody). The method
shows the lowest nonspecific binding while still maintaining high-quality TTR signals. Improved results

HIGH-THROUGHPUT PROTEIN ANALYSIS

53

FIG. 3. High-throughput MSIA analysis of transthyretin and retinol binding protein using human plasma samples from six individuals
randomly arranged in a 96-well titer plate. (A) Mass spectra resulting from the MSIA analysis utilizing anti-TTR derivatized pipette tips.
Shown is the region of the singly charged TTR signals. Highlighted cells show spectra with resolvable parent ion differences between each
other. (B) Mass spectra resulting from the MSIA analysis of the same samples utilizing anti-RBP derivatized pipette tips. Shown is the region
of the doubly charged RBP signals. The highlighted cells represent two spectra with resolvable parent ion differences.

were most likely due to increased functionalization

within the active microflow channels of the frits. Bulk
preparation has a possibility of having dead space volume, leaving large areas unblocked or without Ab coupling, which may lead to higher nonspecifically bound
compounds. Figure 2E shows a spectrum obtained from
an affinity pipette tip prepared in the same manner,
except for the use of anti-RBP antibody. As in Figure

2D, the target protein was sufficiently extracted from

the plasma sample with minimal binding of nonspecific
compounds. As previously mentioned, RBP forms complex with TTR in vivo and it would be expected that
some of the RBP affinity-retrieved in the MSIA tips
would be in the form of an RBPTTR complex. However, it is presumed that the acetonitrile wash performed following the affinity retrieval disrupts the

54

KIERNAN ET AL.

complex, as a result of which TTR signals are not

observed in the resulting mass spectrum. In all, the
in-robotic tips derivatization method was chosen for
the preparation of affinity pipette tips for use in subsequent TTR and RBP screening experiments.
Screening
Plasma/HBS samples from six individuals were aliquotted into wells of a 96-well titer plate, screened in
parallel using anti-TTR affinity pipette tips, and data
were acquired using automated MALDI-TOF MS. Figure 3A shows the results of the screening. The spectra
show a generally high reproducibility, with wild-type
TTR detected in all samples. Mass accuracy for wt-TTR
using external calibration (96 samples: MW (range)
13,74513,787 (0.11% variation)) was reasonable,
considering the dimensions of the titer plate format of
the MALDI target. The same plasma/HBS samples
were next screened using anti-RBP affinity pipette
tips. Due to resolution limitations at higher mass, doubly charged RBP was used for the data analyses. Figure 3B shows the results of the screening. The automated preparation routine was sufficient in producing
samples from which adequate ion signals were obtained with mass accuracies (externally calibrated)
over the surface of the 96-well format target similar to
those observed for TTR (MW (range) 21,040 21,094,
calculated from doubly charged (0.14% variation)).
The resolution of the data (m/dm 800) was sufficient to resolve a second TTR signal in all samples (due
to cysteinylation of Cys10; m 119 Da; (38, 39)) and
a second set of satellite signals at 30 Da higher in
molecular weight than wt-TTR and cys-TTR in 14 replicate analyses of the plasma sample taken from one
individual (Fig. 4A). Such splitting of signals is a general indicator of a point mutation present within the
protein and suggest that one individual in the study
was of heterozygous phenotype, with a genetic polymorphism resulting in a TTR variant shifted in mass
by 30 Da. Regarding RBP, the resolution and quality of the data were sufficient to reproducibly recognize
moderate levels of RBP-Leu (the truncated RBP form)
in all but one of the individuals (individual 4; 15 replicate analyses, Fig. 4B). Currently, there is no clear
definition of the RBP-to-RBP-Leu ratio that is considered normal in humans; however, recent results suggest that an overabundance of RBP-Leu (exceeding the
concentration of RBP) is associated with chronic renal
failure (37). Quantitative assays for both TTR and RBP
are currently under development.
TTR Mapping
Other groups have previously demonstrated the use
of enzymatic mass mapping in combination with

FIG. 4. Phenotypic differences in RBP and TTR within the study

group. (A) Mass spectra resulting from the MSIA analysis of two
samples showing the presence of wild-type and cysteinylated forms
of TTR (wt-TTR and cys-TTR) in both samples and the existence of a
point mutation in one of the individuals (bottom trace), indicated by
the splitting of the two TTR signals. (B) Mass spectra resulting from
the MSIA analysis of two samples showing the presence of only
native RBP in one individual (upper trace) and both native RBP and
RBP-Leu in a second individual (bottom trace). Shown is the region
of the doubly charged RBP signals.

MALDI-TOF MS for the analysis of point mutations

present in TTR (40, 41). Of particular interest is the
work of Theberge et al., who used enzymatically active
MALDI-TOF MS targets in mapping TTR for the presence of mutations (40). In their work, trypsin-activated
gold targets were manufactured as previously described (2123) and applied in the rapid mass mapping
of TTR immunoprecipitated from serum/plasma samples. The approach was shown able to detect the presence of a Val30Met mutation, which is known to be the
cause of familial amyloidic polyneuropathy, the most
common form of amyloidosis (29). Moreover, and fitting
with the work presented here, the authors commented
on the suitability of the enzymatic-target approach to
high-throughput point mutation analyses. Following
suit, TTR from the six individuals participating in this
study were mapped in parallel using a trypsin-active
MALDI-TOF MS target.

HIGH-THROUGHPUT PROTEIN ANALYSIS

55

was present in the mass spectrum (Figs. 5A and 5B,

lower traces). There are two possible (known) point
mutations in this tryptic fragment that would result in
a nominal 30-Da mass shift. Of these, Thr119Met
(m 29.992 Da) agrees most closely with the observed mass difference (versus m 30.011 Da for
the other possible mutation; Ala109Thr). The
Thr119Met variant has been characterized previously
and is referred to as TTR Chicago variant, which is
found to be nonamyloidogenic (42, 43).
CONCLUSION

FIG. 5. Tryptic mapping of MSIA-isolated TTR via utilization of

bioreactive probes. (A) Mass spectra showing two tryptic maps obtained after MSIA analysis of the two samples shown in Fig. 4A. The
inset illustrates a coverage map of the TTR sequence with the observed peptide fragments. (B) Expanded regions of the two mass
spectra showing the exact mass difference between the wt and mutant peptide fragments.

Trypsin digests of TTR from the six individuals were

performed in parallel by eluting the TTR with a low-pH
solution directly from the affinity pipette tips onto
trypsin-activated sites on a bioreactive probe. Following a previously defined protocol (22), the trypsin-active sites were precoated with TrisHCl, which acted
as a compensating buffer, raising the pH of the eluent
(pH 2.5) to that of the working range of trypsin (pH
7 8). The digests were allowed to proceed for 15 min
in a humidified environment (maintained at 37C) before termination by the addition of an acidified MALDI
matrix. Figures 5A and 5B (upper traces) are spectra
representative of the mass maps obtained for five of the
six individuals. The map yielded 80% TTR sequence
coverage (Fig. 5A, inset), which is sufficient to analyze
all but 4 of the 80 known mutations (26). Moreover, the
mapping data are consistent with the presence of only
wt-TTR. Mass maps of the presumably mutated sample demonstrated the same sequence coverage as observed for wt-TTR. However, an additional signal associated with the T12 fragment (at m 29.988 Da)

The use of affinity isolation in combination with

MALDI-TOF MS holds much promise for the detection
of protein variants stemming from genetic, transcriptional, or posttranslational affects. However, in order
to reach its fullest potential, the approach must be
scaled for the analysis of significantly large numbers of
samples, thereby allowing correlations that are able to
statistically differentiate between nondetrimental protein variations and those linked to disease/ailment. We
have reported here progress in the development of a
high-throughput system, composed of parallel robotics
fitted with specialized affinity extraction and sample
processing devices and automated MALDI-TOF MS,
that is capable of assaying specific proteins present in
complex biological media. By addressing multiple samples robotically and in parallel, the time required for
reagent and sample preparation is on the order of 100
samples/h. Data acquisition using automated MALDITOF MS is equally rapid and when performed on native proteins is able to provide a first glance into the
nature of protein variants found in individuals. When
warranted, more detailed structural analyses are possible by digesting proteins enzymatically and analyzing the results using high-performance MALDI-TOF
MS. Given the ability to rapidly perform such analyses,
the high-throughput approach demonstrated here is
likely to find much use in proteomics/clinical studies
where the exceptional structural characterization of
specified proteins present in large populations is required.
ACKNOWLEDGMENTS
This publication was supported in part by Grants 2 R44 GM5660301, 2 R44 GM56580-0, and R01 GM55872-02 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 Institutes of Health.

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