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J. Sep. Sci. 2009, 32, 2937 – 2943
Bjørn Winther1
Marianne Nordlund2
Elisabeth Paus2
Lon Reubsaet1
Trine Grønhaug Halvorsen1
1
Department of Pharmaceutical
Chemistry, School of Pharmacy,
University of Oslo, Oslo, Norway
2
Central Laboratory,
Radiumhospitalet,
Rikshospitalet, Oslo University
Hospital, Oslo, Norway
B. Winther et al.
2937
Original Paper
Immuno-capture as ultimate sample cleanup in LCMS/MS determination of the early stage biomarker
ProGRP
This paper presents a selective and efficient sample preparation procedure for
NLLGLIEAK, signature peptide for the small cell lung cancer (SCLC) biomarker
ProGRP, in human serum. The procedure is based on immuno-capture of ProGRP in
96-wells microtiter plates coated with the mAb E146. After immuno-capture and
thorough rinse, trypsin was added for in-well digestion. Subsequently the signature
peptide was enriched by SPE and determined by LC-MS/MS. Various steps in the procedure were optimized to achieve a low LOD such as dilution of sample, tryptic
digestion, and SPE cleanup and peptide enrichment conditions. A single quadropole
MS was used during optimization of the method. A triple quadropole MS was used
in the method evaluation in order to improve sensitivity. The evaluation showed
good repeatability (RSD, 11.9 – 17.5%), accuracy (3.0 – 6.6%), and linearity (r2 = 0.995)
in the tested range (0.5 – 50 ng/mL). LOD and LOQ were in the pg/mL area (0.20 and
0.33 ng/mL, respectively), enabling the determination of clinically relevant concentrations. The method was applied to two patient samples and showed good agreement with an established immunological reference method. The final method was
compared to a previous published LC-MS method for the determination of ProGRP
in serum based on protein precipitation and online sample cleanup. Both showed
acceptable method performance, however, the immuno-capture LC-MS method was
superior with respect to sensitivity. This illustrates the large potential of immunocapture sample preparation prior to LC-MS in protein biomarker quantification.
Keywords: Immuno-capture / LC-MS/MS / ProGRP / Signature peptide / Tryptic digestion /
Received: April 2, 2009; revised: May 25, 2009; accepted: May 25, 2009
DOI 10.1002/jssc.200900233
1 Introduction
Progastrin releasing peptide (ProGRP) is a protein biomarker, which among other biomarkers, in the case of
increased concentrations indicates small cell lung cancer (SCLC) [1 – 4]. This aggressive disease, with rapidly
growing neoplasm and early metastasis, is highly sensitive to early initiated systemic chemotherapy and radiation. This makes early diagnosis and treatment monitoring crucial.
The reference value for ProGRP in human serum is
l60 pg/mL [5], but the ProGRP concentration can, in the
Correspondence: Dr. Lon Reubsaet, School of Pharmacy, Department of Pharmaceutical Chemistry, University of Oslo, P.O.
Box 1068 Blindern, NO-0316 Oslo, Norway
E-mail: [email protected]
Fax: +47-22854402
Abbreviations: PPT, protein precipitation; ProGRP, progastrin
releasing peptide; RAM, restricted access media; SCLC, small cell
lung cancer
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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
case of SCLC, increase to low and medium ng/mL levels
[2]. Concentration determination is being currently carried out using immunoassay with enzymatic or fluorescence detection [5, 6]. In general, this methodology with
reason accounts for a large amount of protein determinations at low concentrations. With the rapid growing
interest in protein analysis using proteomic approaches,
chromatographic separations coupled to mass spectrometric detection (LC-MS) is an emerging technique in
absolute protein quantification. Although more complex
and less robust compared to the common immunochemical methods, it is less prone to cross-reactivity and has a
competitive sensitivity. It has been demonstrated that
several proteins like prions in brain tissue [7], the kidney
dysfunction marker cystatin C [8], hemoglobin A2 in
whole blood and dried blood spots [9], as well as the
potential serum biomarker for prostate cancer Zn-a2 glycoprotein [10] can be quantitatively determined by this
approach. Determination was achieved by first subjecting the sample to tryptic digestion and then subse-
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B. Winther et al.
quently monitoring tryptic peptide(s) specific for the proteins to be determined. A single specific tryptic peptide
will then be sufficient to determine the equimolar
amount of protein it originates from assuming reproducible digestion. Such a specific tryptic peptide is defined
as a signature peptide.
ProGRP has also been determined using this approach.
In this case, the tryptic peptide NLLGLIEAK was identified
as signature peptide. Concentrations down to 1.5 ng/mL
(l200 fmol/mL) ProGRP in serum could be detected using
acetonitrile (MeCN) induced protein precipitation and
online sample cleanup prior to chromatography.
Although clinically relevant, this LOD is still above the
reference value [11, 12]. Attempts to improve the LOD by
using a triple quadropole mass spectrometer instead of a
single quadropole mass spectrometer only contributed
to a less complex chromatogram but not to a lower LOD.
This was discussed in ref. [12] and explained by possible
ion-suppression, which might be circumvented by better
sample preparation.
The combination of advantages from both immunoaffinity and LC-MS is fronted as the next phase of clinical
applications to determine very low abundant proteins in
complex biological matrices. Two different strategies
have been described using either antibodies for protein
or antibody for proteolytic peptides, both reporting
promising results [13 – 17]. The antibody for protein strategy has one great advantage as these antibodies are often
already available, making the strategy more easily
employable.
In the current paper, the potential of an immuno-capture step in the LC-MS based ProGRP determination was
evaluated. The low reference value of ProGRP has proven
to be a challenge. Even optimized conventional sample
preparation techniques, such as protein precipitation
combined with online restricted access media (PPT-RAM),
have fallen short with respect to sample cleanup and sensitivity [12]. The convenience of available antibodies for
ProGRP [5] made it feasible to investigate immuno-capture as a sample cleanup step prior to tryptic digestion
and LC-MS. A comparison of immuno-capture sample
preparation and the PPT-RAM technique, applied to
ProGRP, is performed with focus on sample cleanup, and
effect on the LOQ. In addition applicability of the
immuno-capture LC-MS method on realistic samples will
be discussed.
2 Experimental
2.1 Chemicals
ProGRP (31 – 98) was produced and patient samples were
provided by the Central Laboratory, Radiumhospitalet,
Rikshospitalet, Oslo University hospital (Oslo, Norway).
All participants had given written informed consent to
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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Sep. Sci. 2009, 32, 2937 – 2943
participate in the study. TPCK treated and lyophilized
sequencing grade trypsin from bovine pancreas was purchased from Sigma – Aldrich (St. Louis, MO, USA) and
human serum samples from healthy subjects were
obtained from Oslo University Hospital, Ullevaal (Oslo,
Norway). All other chemicals used were of analytical
grade.
2.2 Preparation of standards
Stock solutions of ProGRP (31 – 98) were prepared in
100 mM aqueous triethanoleamine (TEA) at pH = 7.3.
These solutions were stored at – 328C and care was taken
to avoid excessive freeze – thaw cycles. The stock solutions were used to spike serum samples for the immunocapture experiments and to prepare the ProGRP in
50 mM freshly prepared ammonium bicarbonate buffer
used in the in-solution digestion experiments. Serum
samples and ammonium bicarbonate buffer were spiked
on the day of the experiments. The volume of spiking
was kept negligible compared to the total volume of sample.
2.3 LC-MS
2.3.1 Single quadropole
The system consisted of a Shimadzu SIL-10ADvp auto
injector, two Shimadzu LC-10ADvp gradient pumps, a
Shimadzu DGU-14A degasser, a Shimadzu SCL-10Avp system controller, and a Shimadzu LCMS-2010A singlequadropole MS detector. Data acquisition and processing
were carried out using Shimadzu LCMS Solution software Version 2.04-H3 (all Bergman, Lillestrøm, Norway).
The ESI source was operated in the positive ionization
mode. The MS was set to monitor the ProGRP-specific
digest peptide NLLGLIEAK [M + 2H]2+ m/z 485.8. The MS
operating conditions were as follows: drying gas between
10 and 20 L/min, nebulizer gas 1.5 L/min, CDL temperature 2008C, block temperature 2008C and probe voltage
+4.5 kV.
2.3.2 Triple quadropole
This system consisted of a Waters 2795 liquid chromatograph, Waters 600 LCD controller and a Waters Quattro
micro-MS/MS detector. System control and data acquisition was performed with MassLynxm version 4.0 SP4 (all
Waters Corporation, Milford, MA, USA). The interface
was ESI operated in the positive ionization mode. The MS
was set to monitor the precursor-fragment ion transition
of the ProGRP-specific digest peptide NLLGLIEAK
[M + 2H]2+ (m/z 486.01) to the y7 ion [M + H]+ (m/z 743.74).
The optimized fragmentation conditions [12] for the
SRM of NLLGLIEAK were with a cone voltage of 25 V and
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J. Sep. Sci. 2009, 32, 2937 – 2943
collision energy at 14 eV. The dwell time was 600 ms
with an inter-scan delay of 100 ms.
2.4 Chromatographic conditions
Chromatographic separation was carried out on a Biobasic-C8 (Teknolab AS, Kolbotn, Norway) column with average pore size 300 , particle diameter 5 lm and column
dimensions 50 mm61 mm id.
The mobile phases consisted of A: 20 mM aqueous formic acid and MeCN (95:5 v/v) and B: 20 mM aqueous formic acid and MeCN (5:95 v/v).
A two-step linear gradient was used. The system was
first kept isocratic at 5% mobile phase B for 1 min after
injection of the sample. The first gradient was then run
from 5% mobile phase B to 20% mobile phase B in 4 min,
and was immediately followed by the second gradient
running from 20 to 30% mobile phase B in 11 min.
Mobile phase B was then increased to 60% in 0.1 min and
kept constant for 2 min before it was returned to starting
conditions in 0.1 min. The column was regenerated with
10 column volumes. Flow rate was set to 50 lL/min and
the injection volume was 20 lL.
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2939
After each step, the flow-through fraction was collected.
All fractions were evaporated to dryness under a stream
of N2 gas at 608C, reconstituted in 25 lL of 20 mM aqueous formic acid and analyzed on the content of
NLLGLIEAK.
2.5.3 Final SPE procedure
Up to 125 lL of sample was applied. After a washing step
(50 lL of 15% MeCN in 20 mM aqueous formic acid) the
peptide was eluted using 50 lL of 45% MeCN in 20 mM
aqueous formic acid. The eluate was evaporated to dryness under a stream of N2 gas at 608C, reconstituted in
25 lL of 20 mM aqueous formic acid and analyzed on the
content of NLLGLIEAK.
2.5.4 Calculation of extraction recoveries
The extraction recoveries were defined as the percentage
of the total analyte amount (originally applied to the SPE
tip), which was recovered after SPE elution, evaporation
to dryness and reconstitution.
2.6 Preparation and use of 96-well immuno plates
2.6.1 Coating of mAb E146 to microtiter plates
2.5 SPE
SPE was carried out by using in-house made SPE tips. All
tips were activated using 100 lL of 95% MeCN in 20 mM
aqueous formic acid and then washed with 100 lL of
20 mM aqueous formic acid prior to use. Each step in the
SPE procedure was carried out by loading the solution on
the top of the tip. In order to press the liquids through
the SPE tip, it was placed on a 10 mL syringe. Pressure
was applied manually.
2.5.1 Preparation of in-house SPE tips
A Pasteur pipette was used to punch six small cushions
(diameter l1 mm) from a 3M Emporem C18 disk (Teknolab AS, Kolbotn, Norway). The C18 material was transferred from the Pasteur pipette to the bottom of a 300 lL
pipette tip (TIP 300 lL Bevel bulk, VWR International,
Oslo, Norway) using a thin metal wire. After transfer, the
six cushions were carefully pressed together in the narrow, lower part of the tip.
2.5.2 Determination of optimal SPE conditions
Optimization of the SPE procedure was performed in
samples digested in-solution (sample volume 300 lL containing 50 ng/mL ProGRP in 50 mM freshly prepared ABC
and 100 ng trypsin, digested o/n at 378C). The optimization was carried out as follows: after application of 50 lL
of digested sample the flow-through of the sample solution from the SPE was collected. Then six 50 lL aliquots
of solutions with increasing amounts of MeCN (0 – 90% in
20 mM aqueous formic acid) were applied to the tip.
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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The mAb E146 was produced by immunizing mice with
recombinant ProGRP (31 – 98) and establishing a hybridoma cell line as described by Nordlund et al. [6].
Immobilization of purified E146 (1 lg/well) was then
performed on Maxisorp microtiter plates (Nunc, Copenhagen, Denmark) as described earlier [18]. In brief, the
antibody solution (1 mg/mL) was first acid treated for
10 min at pH 2.8 by adding six volumes of 100 mM glycine buffer pH 2.5, and then neutralized with 200 mM
sodium dihydrogen phosphate buffer pH 4.3 to an antibody concentration of 5 lg/mL. After dispensing 200 lL
of solution in each well, the microtiter plates were incubated under humidified conditions at 378C for 20 h.
Then the plates were washed twice with Delfia wash solution (50 mM Tris, 150 mM NaCl, 0.1% Germall, and 0.05%
Tween 20 pH 7.8, PerkinElmer Life and Analytical Sciences, Waltham, MA, USA) and incubated with 300 lL/well
of blocking buffer (50 mM Tris, 6% sorbitol, 0.05% azide
pH 7.0) for another 20 h under humidified conditions at
room temperature. The plates were aspirated and kept
dry until use.
2.6.2 Immuno-capture
The immuno-capture method was based on the immunocapture step in the immunofluorometric assay described
in ref. [6]. The samples (fortified serum from healthy subjects and patient samples) were applied to the 96-well
immuno microtiter plates in 200 lL aliquots. When the
effect of sample dilution prior to immuno-capture was
tested, the serum samples were diluted with an aqueous
protein-containing buffer consisting of 50 mM Tris-HCl
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B. Winther et al.
pH 7.8, 150 mM NaCl, 20 lM diethylene triamine pentaacetic acid, 100 mg/L Tween 20, 3 mg/L tartrazine, 1 g/L
Germall, 5 g/L BSA, 1 g/L bovine IgG, and 60 mg/L MAK
33-IgG pPoly [5]. One hundred microliter of this buffer
was added to wells containing 100 lL of serum, thus
diluting the sample twice.
To capture the proteins the plates were subsequently
shaken for 1 h at 500 rpm on a Heidolphm Vibramax
vibrating platform shaker (Kelheim, Germany) to facilitate antigen – antibody interaction. Afterward, the plates
were washed six times with Delfia wash solution using a
Delfia Platewash (both PerkinElmer). Subsequently, the
plates were washed manually twice with 10 mM Tris-HCl
pH 7.4 to ensure removal of Tween and Germall prior to
digestion since these components might interfere with
SPE and LC-MS analysis.
J. Sep. Sci. 2009, 32, 2937 – 2943
Figure 1. Plot of the accumulating amount of NLLGLIEAK
normalized to 100% (lstdev) against the eluting strength in
SPE (n = 3). Extraction recovery, 62%.
After washing, 200 lL of 50 mM freshly prepared ammonium bicarbonate buffer was added to the wells. Tryptic
digestion was carried out by adding 1 lL of freshly prepared trypsin in 50 mM ammonium bicarbonate buffer.
During optimization the trypsin concentration varied
from 0.001 to 10 mg/mL. The wells were sealed using parafilm and placed on the Heidolph Vibramax vibrating
platform shaker and shaken for 5 min at 800 rpm. Following this, the wells were placed in a 378C stove allowing overnight digestion. Up to 125 lL of the sample was
subjected to SPE.
retention. With immuno-capture and in-well digestion,
it is in addition important that the signature peptide is
not a part of the amino acid sequence of the antibodies
and proteins present on the wall of the wells. A BLAST
experiment (http://blast.ncbi.nlm.nih.gov/) for the
sequence NLLGLIEAK in databases for Homo sapiens (tax.
ID 9606), murine (tax. ID 10090), and bovine (tax. ID
9913) showed that in similarity with proteins originating
from humans, none of the proteins used in the immunocapture procedure contained this amino acid sequence.
We could therefore conclude that NLLGLIEAK is a valid
signature peptide for this study.
2.7 Evaluation of the final method
3.2 SPE
To evaluate the potential of immuno-capture, SPE, and
LC-MS/MS analysis of ProGRP, repeatability (n = 5) and
accuracy (n = 3) were tested at 2, 15, and 50 ng/mL
ProGRP in human serum. In addition, the linearity of the
response was evaluated at seven concentrations (n = 2):
0.5, 2, 7, 15, 25, 50, and 100 ng/mL.
LOD and LOQ were estimated from the peak heights of
samples fortified with 0.5 and 2 ng/mL ProGRP in relation to the noise level. S/N of 3:1 was used to estimate
LOD and S/N of 5:1 was used to estimate LOQ.
The method evaluation was performed using the triple
quadrupole.
3.2.1 Application, wash, and elution
2.6.3 In-well digestion
3 Results and discussion
3.1 Selection of signature peptide
As reported in earlier studies [11, 19], NLLGLIEAK can be
used as signature peptide for ProGRP. Selection of the signature peptide and the specificity of this peptide is thoroughly described earlier [11]. In short, this selection was
based on peptide signal intensity, absence of missed
cleavage sites, peptide specificity, and adequate column
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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
To optimize the SPE wash- and elution-steps, three inhouse made SPE tips were used. After sample application
flow-through fractions from each elution-step with
increasing amounts of MeCN in 20 mM aqueous formic
acid were determined on their content NLLGLIEAK. Figure 1 shows the cumulative relative amount of
NLLGLIEAK plotted against the MeCN content of the eluent. The cumulative amount was determined by summing the signal intensities of each elution step with the
signal intensity of all prior elution steps for the individual SPE tip. From Fig. 1 it is clear that washing can be performed using 20 mM aqueous formic acid containing
15% MeCN without loosing NLLGLIEAK, while 30% MeCN
results in virtually complete elution. To ensure a robust
system with complete elution of NLLGLIEAK from the
SPE column, eluent containing 45% MeCN in 20 mM
aqueous formic acid was used for the remainder of the
studies.
3.2.2 Recovery of SPE after in-well digestion
After optimization of the SPE wash and elution conditions the final conditions were applied to enrich an inwww.jss-journal.com
J. Sep. Sci. 2009, 32, 2937 – 2943
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2941
Figure 2. Plot of NLLGLIEAK intensity against the amount of
trypsin added (n = 2).
well digest of 25 ng/mL ProGRP in serum after immunocapture. Extraction recovery was determined to 65%
(n = 2; average peak height prior to SPE, 5242; average
peak height after SPE, 16 963; preconcentration factor
(125 lL ﬁ 25 lL), five times). Linearity up to 50 ng/mL, as
shown later, indicates that this value for recovery is
achieved at all tested concentration levels.
Figure 3. Single MS chromatogram (SIM at m/z = 485.8) of
the signature peptide for (a) undiluted serum and (b) diluted
serum after immuno-capture, in-well digestion and SPE.
Both samples were fortified with 1 ng/mL ProGRP.
3.3 Evaluation of the in-well digestion
3.3.1 Effect of trypsin concentration on the in-well
digestion
The amount of trypsin to be used is related to the amount
of protein present. In an in-solution digest this ratio
varies from l1:25 (w/w) to lower ratios. In the case of inwell digestion, it is difficult to estimate the total protein
amount since in addition to the protein of interest both
antibodies and other proteins utilized during preparation and immuno-capture will be present in the well during digestion. To circumvent this challenge the effect of
the amount of trypsin added was investigated. Figure 2
shows the intensity of the signature peptide plotted
against the amount of trypsin added. The lowest trypsin
amount added to the wells, which gave good digest with
high NLLGLIEAK formation, was 100 ng. This amount
was achieved by adding 1 lL of 100 lg/mL trypsin to each
well, and was used throughout the study.
3.3.2 Dilution of the samples
In immunoaffinity assays, serum samples are often subjected to dilution prior to application on the microtiter
plates. In order to investigate the possible positive effects
of serum dilution during immuno-capture, a comparison
was made between undiluted serum and serum diluted
1:1 with a protein-containing buffer, as described in Section 2. Figure 3 shows the typical SIM-traces (m/z = 485.8)
of LC-MS (single-quadropole) analyses of both undiluted
and diluted serum. It is clear that the undiluted serum
(Fig. 3a) yields higher peak intensities for both the signa-
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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ture peptide and impurities compared to that of the
diluted serum (Fig. 3b). The noise level was similar in
both analyses, hence, highest S/N was obtained for undiluted serum. It was, therefore, decided to use undiluted
serum for the subsequent experiments.
3.4 Evaluation of the method
3.4.1 Method performance
The final method was validated using an LC-MS/MS (triple
quadrupole) for increased sensitivity. Optimal SRM conditions had been determined previously [12]. Repeatability, accuracy, linearity and LOD, LOQ were determined
for the ProGRP signature peptide NLLGLIEAK in human
serum after immuno-capture, in-well digestion and SPE.
Table 1 shows the results for repetitive extractions at
three different ProGRP concentrations. RSDs were similar at low, medium, and high ProGRP concentrations.
Table 2 shows the values for accuracy determined at
three concentrations of ProGRP. These values were also
independent of the concentration of ProGRP analyzed.
A calibration curve was constructed from 0.5 to
100 ng/mL ProGRP in human serum. A quadratic polynomial curvature (y = – 0.6709x2 + 213.25x + 83.095) fitted
concentrations up to 100 ng/mL well (r2 = 0.997). However, as apparent linearity was observed between 0.5 and
50 ng/mL ProGRP (y = 182.1x + 226, r2 = 0.995), it was
decided to evaluate the typical validation parameters for
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B. Winther et al.
J. Sep. Sci. 2009, 32, 2937 – 2943
Figure 4. Triple quadrupole ionextract of the SRM transition
486.01 ﬁ 743.74 of (a) blank
human serum, (b) human serum
from subject 1, and (c) human
serum from subject 2. The signal
intensity of the NLLGLIEAK fragment (m/z = 743.74) at
tr = 17.07 min in (c) equals 100%
signal intensity.
Table 1. Within-day repeatability of ProGRP from human
serum samples (n = 5)
Spiked ProGRP
conc. (ng/mL)
Signal intensity
RSD (%)
NLLGLIEAKa) (average l SD)
2
15
50
564 l 70
3295 l 576
9647 l 1144
12.4
17.5
11.9
Table 3. Analysis of patient samples
Subject 1
Subject 2
ProGRP immuno- ProGRP immunoMS (ng/mL)
fluorometric
assaya) (ng/mL)
Bias
(%)
0.72
26.62
– 29
15
1.01
23.24
a)
a)
SRM transition: 486.01 ﬁ 743.74.
Table 2. Accuracy of ProGRP from human serum samples
(n = 3)
Spiked ProGRP
conc. (ng/mL)
Determined ProGRP
conc. (ng/mL)
Accuracy
(%)
2
15
50
2.1
15.4
52.5
6.6
3.0
5.1
concentrations up to 50 ng/mL ProGRP in human serum
using linear calibration.
The LOD and LOQ were 0.2 and 0.33 ng/mL ProGRP in
human serum, respectively. The evaluation shows acceptable repeatability, accuracy, and linearity. The LOD and
LOQ were in the pg/mL area, which is of clinical relevance.
3.4.2 Analysis of patient samples
The method was applied to the analysis of serum from
two subjects with elevated serum-ProGRP level. Table 3
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2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Described by Nordlund et al. [5], and regarded as reference method as it is used on routine basis for analyzing
patient samples.
Comparison with established immunofluorometric assay.
shows the average ProGRP concentration (n = 2) of the
two subjects determined both by the established immunofluorometric assay (described by Nordlund et al. [5])
and with the immuno-capture LC-MS/MS method. Serum
levels of ProGRP assayed by the two procedures were
comparable, indicating that immuno-capture LC-MS/MS
has a potential in ProGRP quantification. However, the
LOQ (16 pg/mL) of the immunofluorometric assay is still
superior to that of the present immuno-capture method.
Figure 4 shows the ion-extracts of the SRM transition
486.01 ﬁ 743.74 for a blank human serum sample as
well as for subjects 1 and 2.
3.4.3 Method comparison
The analytical performance of the immuno-capture
method was compared with the performance of the previously described LC-MS method utilizing protein precipitation and online sample cleanup (PPT-RAM method
[12]). The LOD improved with a factor of 7.5 utilizing the
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J. Sep. Sci. 2009, 32, 2937 – 2943
immuno-capture method. This improvement was
achieved despite a five-fold reduction in the sample volume (from 1 to 0.2 mL), illustrating the large potential of
combining immuno-capture and LC-MS/MS. Both the
immuno-capture method and the PPT-RAM method
showed acceptable and comparable repeatability and calibration performance. While the accuracy of the two
methods was comparable at higher ProGRP concentrations the accuracy of the immuno-capture method was
superior to the PPT-RAM method in the lower range
(accuracy of 22% at 5 ng/mL vs. 6.6% at 2 ng/mL, respectively). However, contrary to the PPT-RAM method the
values for the immuno-capture method was obtained
without the use of internal standard, indicating the
potential of even better method performance by applying internal standard for quantification.
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2943
any of these strategies is chosen a more thorough validation including the use of internal standard should be
employed in order to ensure reliable quantification of
the biomarker(s).
The authors wish to thank Niclas Lunder from the Department of
Psychopharmacology, Diakonhjemmet Hospital, Oslo, Norway for
his valuable contribution to the analysis on the triple quadrupole
MS/MS.
The authors declared no conflict of interest.
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4 Concluding remarks
The present work is the first report on an LC-MS based
ProGRP analysis using a selective immuno-capture step.
Selective immuno-capture of ProGRP in 96-wells microtiter plates followed by in-well digestion and subsequent
SPE enrichment of the signature peptide prior to LC-MS/
MS analysis resulted in efficient sample cleanup and
enrichment from human serum. Good repeatability,
accuracy, and linearity were seen in the tested range. In
addition, LOD and LOQ were in the pg/mL area, enabling
reliable determination of clinical relevant concentrations. Two patient samples were analyzed and the
method showed good agreement with an established
immunological assay.
A comparison with an LC-MS method for ProGRP utilizing conventional sample preparation methods demonstrated the potential of immuno-capture sample pretreatment especially with respect to sensitivity.
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improved even further. This can possibly be done by
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