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Clinical Chemistry 51: 973-980, 2005. First published April 21, 2005; 10.1373/clinchem.2004.047308
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Right arrow Proteomics and Protein Markers
(Clinical Chemistry. 2005;51:973-980.)
© 2005 American Association for Clinical Chemistry, Inc.

Proteomics and Protein Markers

Standardized Approach to Proteome Profiling of Human Serum Based on Magnetic Bead Separation and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Sven Baumann2, Uta Ceglarek2, Georg Martin Fiedler, Jan Lembcke, Alexander Leichtle and Joachim Thierya

Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital, Leipzig, Germany.

aAddress correspondence to this author at: Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Liebigstrasse 27, 04103 Leipzig, Germany. Fax 49-341-9722209; e-mail thiery{at}medizin.uni-leipzig.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Magnetic bead purification for the analysis of low-abundance proteins in body fluids facilitates the identification of potential new biomarkers by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The aims of our study were to establish a proteome fractionation technique and to validate a standardized blood sampling, processing, and storage procedure for proteomic pattern analysis.

Methods: We used magnetic bead separation for proteome profiling of human blood by MALDI-TOF MS (mass range, 1000–10 000 Da) and studied the effects on the quality and reproducibility of the proteome analysis of anticoagulants, blood clotting, time and temperature of sample storage, and the number of freeze–thaw cycles of samples.

Results: The proteome pattern of human serum was characterized by ~350 signals in the mass range of 1000–10 000 Da. The proteome profile showed time-dependent dynamic changes before and after centrifugation of the blood samples. Serum mass patterns differed between native samples and samples frozen once. The best reproducibility of proteomic patterns was with a single thawing of frozen serum samples.

Conclusion: Application of the standardized preanalytical blood sampling and storage procedure in combination with magnetic bead-based fractionation decreases variability of proteome patterns in human serum assessed by MALDI-TOF MS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Proteomic pattern analysis by mass spectrometry (MS) 1 is one of the most promising new approaches for the identification of potential blood biomarkers to assess health and disease (1)(2). The discovery of biomarkers in body fluids has been advanced by the recent introduction of MS-based screening methods such as surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) MS and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS (3).

Techniques such as 2-dimensional gel electrophoresis and multidimensional liquid chromatography after MS analysis can remove signal-suppressing components of complex biological mixtures such as serum or cell extracts (4)(5)(6)(7)(8), but these techniques are laborious and time-consuming. In a new pretreatment method, serum peptides are fractionated and concentrated on surface-modified targets with specific protein-capture properties. These targets can be used for TOF MS directly (SELDI-TOF MS) (9). The first clinical investigations using SELDI-TOF MS for different cancer types (e.g., ovarian, prostate, bladder, breast, lung, and brain) revealed high diagnostic sensitivities and specificities (10)(11)(12)(13)(14)(15)(16). However, for the same cancers, different patterns were identified by various research groups using the same types of biological specimens and the same analytical platform (17). These discrepancies could be attributable to poorly defined preanalytical protocols. For accurate MS analysis, the proteome fractionation procedure and the preanalytical conditions for proteome mapping must be carefully assessed (18).

The aim of this study was to establish a well-defined protocol for precise proteome analysis and preanalytical blood sample collection, storage, and processing procedures to minimize the variability in data obtained for the proteome pattern. We used a novel sample pretreatment procedure with surface-modified magnetic beads before identification of human serum proteome profiles using MALDI-TOF MS (19)(20).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
chemicals and calibrators
Gradient-grade acetonitrile and ethanol were obtained from J.T. Baker; p.a. trifluoroacetic acid and acetone were purchased from Sigma-Aldrich. The peptide calibrator containing angiotensin II, the protein calibrator, and {alpha}-cyano-4-hydroxycinnamic acid were purchased from Bruker Daltonics GmbH. For magnetic bead preparations, we used 0.2-mL polypropylene tubes (8-tube strips) from Biozym. Multifly needle sets and polypropylene Monovettes with and without anticoagulants (EDTA, heparin, and citrate) were obtained from Sarstedt.

blood samples
Blood samples from 20 healthy volunteers (10 females and 10 males; age range, 26–39 years) were collected in serum, EDTA, heparin, and citrate Monovettes. Serum and plasma aliquots were stored in 1.5-mL polypropylene tubes (NeoLab). The blood samples were processed according to a standardized protocol. After sample collection, the Monovettes were incubated at room temperature (25 °C) for 30 min and centrifuged at 1400g for 10 min. The plasma and serum samples were immediately frozen in aliquots of 100 µL at –80 °C; for proteome fractionation, samples were thawed at room temperature for 15 min and processed immediately. Variations in sample pretreatment are described separately below. For precision and mass accuracy analyses, serum and plasma samples were pooled from 20 individual samples (from 10 females and 10 males).

proteome fractionation
Proteome fractionation of the samples was performed with ClinProt purification reagent sets from Bruker Daltonics. Magnetic particles (particle size <1 µm) with defined hydrophobic surface functionalities [magnetic bead–hydrophobic interaction chromatography (MB-HIC) C3, C8, and C18], a mean pore size of 40 nm, and a specific surface area of 100 cm2/g were used.

As recommended in the manufacturer’s protocol, we diluted 5 µL of sample with 10 µL of a binding solution added to the bead slurry (5 µL) in a 0.2-mL polypropylene tube, mixed thoroughly, and incubated the tube for 1 min. After magnetic bead separation and 3 washings, we eluted the proteome fraction from the magnetic beads with 5 µL of an acetonitrile–water mixture (1:1 by volume). We prepared targets (co-crystallization) by spotting 1 µL of a mixture containing 10 µL of 0.3 g/L {alpha}-cyano-4-hydroxycinnamic acid in ethanol–acetone (2:1 by volume) and 1 µL of the eluted proteome fraction on the AnchorChipTM target (Bruker Daltonics).

ms
For the proteome analysis, we used a linear MALDI-TOF mass spectrometer (Autoflex; Bruker Daltonics) with the following settings: ion source 1, 20 kV; ion source 2, 18.50 kV; lens, 9.00 kV; pulsed ion extraction, 120 ns; nitrogen pressure, 2.5 x 105 Pa. Ionization was achieved by irradiation with a nitrogen laser ({lambda} = 337 nm) operating at 50 Hz. For matrix suppression, we used a high gating factor with signal suppression up to 500 Da. Mass spectra were detected in linear positive mode. Mass calibration was performed with the calibration mixture of peptides and proteins in a mass range of 1000–12 000 Da. We measured 4 MALDI preparations (MALDI spots) from each magnetic bead fraction. For each MALDI spot, 300 spectra were acquired (30 laser shots at 10 different spot positions). To increase the detection sensitivity, we removed excess matrix with 6 shots at a laser power of 45% before data acquisition at 25%. All signals with a signal-to-noise (S/N) ratio >3 in a mass range of 1000–10 000 Da were recorded with use of the AutoXecute tool of the flexControl acquisition software (Ver. 2.0; Bruker Daltonics). We used the ClinProTools bioinformatics software (Ver. 1.0; Bruker Daltonics) for proteome pattern recognition.

evaluation of the reproducibility of proteomic profiling using ms
To investigate the efficiency and recovery of magnetic bead fractionation, we mixed the peptide calibration solution containing angiotensin II (75 µg/L) with 40 g/L albumin and with pooled serum. The MALDI-TOF MS (S/N ratio >3) detection limits were determined for analysis of angiotensin II directly and after magnetic bead fractionation using MB-HIC C3, C8, and C18 beads. To evaluate the analytical recovery and variability, we analyzed 10 bead preparations of the peptide/protein calibration mixture and the pooled serum samples on 1 day (within-day variability) and on 3 consecutive days (day-to-day variability), using MB-HIC C3, C8, and C18 beads. To determine the variation in the m/z ratios, we compared relative and absolute peak intensities of 11 selected signals of the pool serum with the 11 signals obtained from the peptide/protein calibration mixture.

evaluation of preanalytical conditions of blood sampling and storage
The proteomic patterns of serum and plasma (EDTA, citrate, and heparin) were investigated in the pooled serum and plasma samples after fractionation with MB-HIC C3, C8, and C18 beads. We investigated the influence of the blood collection tube material (polymeric polypropylene) by incubating the Monovettes with 3 mL of an aqueous solution containing 0.1 mol/L NaCl for 2 h at room temperature with gentle rocking.

To investigate the influence of the blood clotting on the proteomic pattern, we incubated single blood samples at room temperature (25 °C) for 10, 30, 60, 120, and 180 min before centrifugation. One aliquot was immediately prepared after each time point for MALDI-TOF MS; the other aliquots were kept frozen at –80 °C until analysis. The effects of 5 freeze–thaw cycles on the proteomic pattern were investigated.

The effect of time and temperature on the proteomic pattern was investigated in the pooled serum samples. After centrifugation according to the standard protocol, serum aliquots were incubated at 4, 25, and 40 °C for 30, 60, 120, and 240 min each. The samples were divided into 2 aliquots; 1 aliquot was immediately fractionated and analyzed by MALDI-TOF MS and the other was frozen at –80 °C and analyzed 1 week later.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
reproducibility and mass accuracy of maldi-tof ms
In the blood samples, magnetic bead fractionation using MB-HIC C3 (Fig. 1B ), C8 (Fig. 1C ), and C18 (Fig. 1D ) beads allowed identification of ~350 signals with a S/N ratio >3. Direct analysis of the pooled serum sample gave MALDI-TOF MS spectra without discrete mass signals (Fig. 1A ).



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  		Figure 1. Comparison of serum proteome patterns.

(A), direct analysis; (B), fractionation with MB-HIC C3; (C), fractionation with MB-HIC C8; (D), fractionation with MB-HIC C18.

The reproducibility of mass spectrum generation was determined with respect to the relative peak intensities. Within-day CVs for direct MS analysis of the peptide/protein calibration mixture (without magnetic bead fractionation) were 4%–22%, and between-day CVs were 5%–23% (data not shown). Shown in Table 1 are the within- and between-day CVs of the relative peak intensities for 11 signals of the peptide/protein calibration mixture and of the pooled serum samples representing low-, medium-, and high-abundance peptides over the whole mass range after magnetic bead fractionation with MB-HIC C3, C8, and C18 beads. Signals with relative signal intensities >2% gave within-day CVs <14% and between-day CVs <26%.


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  		Table 1. Relative intensities and precision of 11 signals from the peptide/protein calibration solution and serum pool (n = 10) in the mass range 1000–10 000 Da, obtained by MALDI-TOF MS after magnetic bead preparation.

We evaluated the mass accuracy of the MS measurements with 10 magnetic bead preparations (MB-HIC C3, C8, and C18) of the peptide/protein calibration mixture and the pooled serum sample, measured in 1 run and on 3 consecutive days. Maximum mass shifts over the range 1000–10 000 Da were 0.028% for the peptide/protein calibration mixture and 0.035% for the pooled serum sample. These results correlate with a variation of <1 Da in the absolute mass shift for signals <3500 Da.

The detection limit for angiotensin II after direct analysis of the peptide/protein calibration mixture was 6.8 ng/L. After magnetic bead fractionation (MB-HIC C3, C8, and C18), the detection limit for angiotensin II in the calibration mixture containing 40 g/L albumin was 36 ng/L. The detection limit increased to 2000 ng/L for angiotensin II after magnetic bead preparation of the pooled serum with angiotensin II added.

evaluation of preanalytical processing of blood sampling and storage
Compared with EDTA-, heparin-, and citrate-plasma samples (Fig. 2 , A1–C2), the MS spectrum of serum (Fig. 2 , D1 and D2) showed the highest number of signals with the highest intensities. MS spectra of the plasma samples were dominated by signals at 2380.2, 3317.0, and 4713.0 Da (signals not shown). In addition, several signals (e.g., 6631.6 and 9133.8 Da) were observed in plasma and serum. The mass spectrum of heparin plasma was characterized by various signals in the small mass range 1000–2000 Da. The number of signals in the mass spectra increased in platelet-rich citrate plasma (centrifugation at 100g for 10 min; data not shown).



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  		Figure 2. Mass spectra of plasma obtained with different anticoagulants after MB-HIC C18 bead fractionation and MALDI-TOF analysis.

(A1 and A2), citrate; (B1 and B2), EDTA; (C1 and C2), heparin; (D1 and D2), serum.

Interferences from blood collection tube polymeric polypropylene materials (EDTA, heparin, and citrate tubes) were excluded. After magnetic bead separation, no interfering signals with S/N ratios >3 were obtained in the MALDI-TOF mass spectra in the mass range 1000–10 000 Da (data not shown).

The relative intensities of the serum proteome signals changed with time after clotting (Fig. 3 ). A loss of intensity with time was observed for low–molecular-mass signals (e.g., 1352.3 Da). Signals with masses >4000 Da (e.g., 4211.1 Da) increased in relative intensity with time, but signals with lower masses (e.g., 3264.2 Da) did not change in relative intensity. In samples allowed to clot for >120 min at room temperature, the background noise increased considerably (mass range 1000–2000 Da).



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  		Figure 3. Variation in relative intensities for 3 characteristic signals of the pool serum sample after MB-HIC C18 fractionation depending on clotting time.

We compared the mass spectra of unfrozen serum aliquots with the spectra of the same samples after freezing. Unfrozen serum showed a high degree of variation in relative signal intensities, and reproducibility was poor. As shown in Fig. 4 , freezing–thawing markedly decreased the intensities of proteins/peptides at 1538.5, 1554.5, and 1563.4 Da (panel A1, no freezing; panel B1, 1 freeze–thaw cycle; panel C1, 3 freeze–thaw cycles; panel D1, 5 freeze–thaw cycles). The same decrease was observed for signals in the mass range 3200–3300 Da (data not shown). Freezing–thawing did not affect signals for proteins/peptides <1500 Da (e.g., 1467.3 Da). In contrast, signals above 8900 Da (e.g., 8916.3, 9133.8, and 9423.8 Da) increased in relative peak intensity after only 1 freeze–thaw cycle (Fig. 4A2 ). Recurrent freeze–thaw cycles further decreased the intensity of low–molecular-mass signals (e.g., 1538.5, 1554.5, and 1563.4 Da) as shown in panels B1, C1, and D1 of Fig. 4 . However, proteins/peptides with high molecular masses (8916.3, 9133.8, and 9423.8 Da) were remarkably stable and were not influenced by recurrent freeze–thaw cycles (Fig. 4 , panel B2, 1 freeze–thaw cycle; panel C2, 3 freeze–thaw cycles; panel D2, 5 freeze–thaw cycles).



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  		Figure 4. Influence of freeze–thaw cycles on the signal intensities in the pooled serum after MB-HIC C18 bead fractionation and MALDI-TOF analysis.

(A1 and A2), unfrozen; (B1 and B2), 1 freeze–thaw cycle; (C1 and C2), 3 freeze–thaw cycles; (D1 and D2), 5 freeze–thaw cycles.

The influence of time and temperature on the proteomic pattern is shown Fig. 5 . The pattern was stable and showed little degradation after 240 min at 4 °C and did not change for up to 120 min at 25 °C. At 40 °C, however, there was significant loss of intensity for all signals in the proteome profile after only 30 min. During a 7-day storage period at 4 °C, the relative peak intensities and the respective S/N ratios decreased after 24 h for masses <2000 Da. Signals at 3193.2 and 3264.2 Da were also affected. In contrast, we observed no change for signals with a molecular mass >4000 Da.



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  		Figure 5. Influence of storage temperature on signal intensities after MB-HIC C18 bead fractionation and MALDI-TOF analysis of the pooled serum sample.

(A), treated according to the standard protocol; (B), 240 min at 4 °C; (C), 240 min at 25 °C; (D), 240 min at 40 °C.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Protein profiling is a promising tool for the discovery and subsequent identification of proteins and peptides associated with various diseases (2)(21). However, knowledge is still limited regarding the sensitivities and reproducibilities of various techniques for analyzing the wide dynamic range of peptides and proteins in blood (18). The aim of our study was to establish a valid, standardized approach to preanalytical blood sample processing. We found that protein profiling by MALDI-TOF MS after proteome fractionation with magnetic beads is a robust, precise, and rapid technique for the investigation of complex blood samples.

We found ~350 signals in human serum in the mass range 1000–10 000 Da. Previous studies identified several peptides and protein fragments by use of electrospray ionization tandem MS and MALDI-TOF, and these methods may help to assign probable identities to the proteins in our spectra. Plasma protein fragments at m/z 2428 (albumin, 25–45) and 2672 (fibrinogen A{alpha}, 600–624) were identified in human hemofiltrate signals after ion-exchange chromatography and MALDI-TOF MS by Richter et al. (22). Bondarenko et al. (23) investigated the potential of MALDI and electrospray ionization MS for screening of polymorphisms in the apolipoproteins associated with VLDL after solid-phase extraction (C18 modification) of the isolated and delipidated lipoproteins. They identified the major apolipoprotein C (apoC) classes in the VLDL (apoC-I', m/z 6432.4; apoC-I, m/z 6630.6; apo proCII, m/z 8914.9; and apoC-III1, m/z 9421.3). We detected signals with the same m/z ratios in our human serum samples (Fig. 1Up , B–D; Fig. 2Up , D2).

The variability of relative signal intensities in our study was in the range of the previously published data from Zhang et al. (19). They selected 10 high-, medium-, and low-abundance signals in the mass range 1000–10 000 Da and calculated that the variability of each peak area was 11%–25% (19). The intraassay variability in our study was 3%–23%, and interassay variability was 6%–33%. The higher reproducibility in our study was achieved by 4-fold MALDI spotting of each magnetic bead fractionation product. We determined mass accuracy by analyzing serum samples after magnetic bead fractionation on 1 day (n = 10) and on 3 consecutive days. The mean mass shift of 0.035% led to absolute mass differences between 0.35 and 3.5 Da in the mass range 1000–10 000 Da. These absolute mass differences are in accordance with the known mass accuracy of MALDI-TOF MS (24). We solved the problem of mass shift by using the most prominent peaks to recalibrate the spectra with the ClinProTools bioinformatics software (19). These findings support the robustness and precision of the magnetic-bead–based pretreatment technology. Semmes et al. (25) investigated the mass accuracy and reproducibility of serum protein profiling, using SELDI-TOF MS in an interlaboratory study (n = 6). They found mean intra- and interlaboratory mass accuracy differences of 0.1% and a 15%–36% variation in relative signal intensities for the 3 most prominent mass signals with S/N ratios >40. Maximum interlaboratory mass differences of 28 Da were obtained for a mean mass signal of 9297 Da.

The high sensitivity and reproducibility of the novel magnetic-bead–based platform for proteome profiling in our study is supported by a recent study from Villanueva et al. (20). They reported that the use of porous particles for sample pretreatment is more sensitive than surface capture on chips because spherical particles have larger combined surface areas than small-diameter spots. Using the magnetic bead technology, they found 400 different proteome signals in the mass range 800–15 000 Da in serum. For our study, we used comparable beads with the same specifications, but the mass range was smaller (1000–10 000 Da) and produced 350 proteome signals. In our study, because of the high adsorptive capacity of the magnetic beads, the detection limit for the low–molecular-mass peptide angiotensin II in serum was 2000 ng/L after magnetic bead fractionation. The different sensitivities for the detection of angiotensin II added to serum and to aqueous albumin solution could be caused by competition effects during the magnetic bead fractionation of additional low– and high–molecular-mass components in serum. In addition, ionization suppression effects of other serum components may influence the ionization of angiotensin II added to serum. Despite these limitations, the application of magnetic bead fractionation in combination with MALDI-TOF is appropriate for the detection of low concentrations of proteins and peptides in serum (26).

Use of plasma entails the possibility of residual platelet contamination, which could influence the mass spectrum. To achieve reproducible proteome profiling and accurate data interpretation, we evaluated preanalytical conditions of blood sampling and storage (18). We found no interfering signals from the polymeric polypropylene materials in the blood collection tubes (EDTA, heparin, citrate, and serum tubes) in the mass range 1000–10 000 Da. In contrast, Drake et al. (27) described interfering signals in the mass range 1000–3000 Da from polymeric materials in blood collection tubes. In the present study, however, we used a blood collection system from a different manufacturer and a different sample pretreatment procedure.

Our study results showed that anticoagulants, temperature, and freeze–thaw cycles have a significant impact on dynamic alterations of the serum proteome in the mass range 1000–10 000 Da. In a recent study, Schaub et al. (28) investigated preanalytical factors that can modify urine proteome profiling by SELDI-TOF MS. They studied the influence of extrinsic (instrument set-up, storage, freeze–thaw cycles) and intrinsic factors (blood in urine, urine dilution, first void vs midstream urine) on urine values. They found that up to 4 freeze–thaw cycles with freezing at –70 °C did not alter the urine protein profile but that variation in intrinsic factors caused significant differences. Urine, however, is characterized by high amounts of polar low–molecular-mass components and an excess salt content; therefore, direct comparison of preanalytical variables for urine and blood samples is limited.

Proteome profiling using magnetic bead-based proteome fractionation MALDI-TOF MS is a promising screening tool for the identification of specific proteome patterns. It is difficult, however, to identify the proteins and peptides or to quantify the interesting analytes directly by MALDI-TOF MS technology (17). The relative peak intensities can be influenced by ion suppression effects and do not necessarily reflect the concentrations of the detected proteins or peptides. Therefore, additional specific chromatographic separation techniques and mass spectrometric platforms (e.g., liquid chromatography with electrospray ionization tandem MS) are necessary for the further identification and quantification of potential biomarkers (4)(7).

Our results show that magnetic bead fractionation in combination with MALDI-TOF MS is a highly sensitive and reproducible analytical platform for proteome profiling of human blood in the mass range 1000–10 000 Da. Use of our evaluated procedure could reduce preanalytical variables; the procedure involves (a) blood sample collection in polypropylene tubes; (b) incubation of the tubes at room temperature for 30 min to allow coagulation; (c) serum separation by centrifugation (10 min at 1400g) at room temperature; (d) separation of 100-µL aliquots in small polypropylene tubes for immediate freezing at –80 °C (no changes in the proteome pattern were observed for a storage period of at least 6 months); and (e) thawing of frozen aliquots for 15 min at room temperature and immediate processing by MB-HIC proteome fractionation.


   Acknowledgments
 
This work was supported by a grant from the Sächsische Aufbaubank (SAB) and by a grant of the Deutsche Forschungsgemeinschaft, Th 374/2-3. We thank Drs. Markus Kostrzewa and Thomas Elssner (Bruker Daltonics, Leipzig, Germany) for helpful advice and discussions.


   Footnotes
 
1 Nonstandard abbreviations: MS, mass spectrometry; SELDI-TOF, surface-enhanced laser desorption/ionization time of flight; MALDI, matrix-assisted laser desorption/ionization; MB-HIC, magnetic bead–hydrophobic interaction chromatography; S/N, signal-to-noise; and apo, apolipoprotein.

2 These authors contributed equally to this work.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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