Evaluation of the Combination of Bead Technology with SELDI-TOF

Aug 9, 2008 - The bead technology is easily compatible with both SELDI-TOF-MS and 2-D DIGE and the samples can be analyzed directly without any ...
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Evaluation of the Combination of Bead Technology with SELDI-TOF-MS and 2-D DIGE for Detection of Plasma Proteins Carina Sihlbom, Ida Kanmert, Helena von Bahr, and Pia Davidsson* AstraZeneca R&D Mo¨lndal, S-431 83 Mo¨lndal, Sweden Received January 31, 2008

Abstract: Today biomarker discovery is one of the most active aspects of proteomic investigations. However, the wide dynamic range of plasma proteins makes the analysis very challenging because high abundance proteins tend to mask those of lower abundance. Using a large bead-based library of combinatorial peptide ligands (Equalizer beads or ProteoMiner), the dynamic range of the protein concentration is compressed, the high abundance proteins present in the sample are reduced and the low abundance proteins are enriched, while retaining representatives of all proteins within the sample. In the present study, the combination of beads with surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and two-dimensional differential gel electrophoresis (2-D DIGE) technology were evaluated considering efficiency, reproducibility, sensitivity, and compatibility. The bead technology is easily compatible with both SELDI-TOF-MS and 2-D DIGE and the samples can be analyzed directly without any processing of the sample. The use of the beads prior SELDITOF-MS and 2-D DIGE enabled detection of many new protein spots/peaks and increased resolution and improved intensity of low abundance proteins in a reproducible fashion compared with the depletion technique. Several proteins have been identified by the combination of beads, 2-D DIGE and MS for example different kinds of complement factors and cytoskeletal proteins. Our data suggest that integration of the bead technology with our current proteomic technologies will enhance the possibility to deliver new peptide/protein biomarker candidates in our projects. Keywords: bead-based technology • SELDI-TOF-MS • 2-D DIGE • mass spectrometry • plasma proteins

Materials and Methods

Introduction Biomarker discovery in biological fluids such as plasma, urine, or cerebrospinal fluid is often limited by the availability of sufficient volumes and complicated by the wide dynamic range of the human proteome. Potential disease biomarkers are often present at low concentration and a prefractionation method such as depletion, liquid-phase isoelectric focusing * To whom correspondence should be addressed. E-mail: pia.davidsson@ astrazeneca.com. Tel: +46 31 7064146. 10.1021/pr800340c CCC: $40.75

(IEF), or chromatography prior protein profiling can thereby assist in the biomarker process. No single proteomic technique is capable of visualizing every component of a proteome, and compromises are always required. The most common prefractionation method is immunodepletion, which has extensively been used for specific removal of high abundance proteins either based on dye-ligands1,2 or specific antibodies.2–4 However, a number of problems have been outlined, particularly due to codepletion, which might subtract several minor species together with the target proteins. Many of the methods are lowthroughput and tend to dilute the sample, making downstream analysis more challenging. A novel approach for mining the “unseen proteome” is the bead-based library of combinatorial peptide ligands, Equalizer beads or ProteoMiner, recently described by Righetti et al.5 The basic article, outlining the synthesis of the “Equalizer beads” and some of the most important properties, has previously been presented,6 together with several reviews.7,8 The use of Equalizer beads reduces the dynamic range of the proteome to simultaneously dilute high abundance proteins and concentrate low abundance proteins. The eluted material will therefore consists of significantly lower amount of total protein representing a higher diversity of species. Some applications on urine, plasma as well as amplifying impurities on r-DNA products have previously been described.9–11 Only one study has used the bead technology in combination with surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) in a patient population that is nonsmoking patients with lung-cancer.12 The aim of the present study was to evaluate the combination of beads with SELDI-TOF-MS and two-dimensional differential gel electrophoresis (2-D DIGE) regarding their efficiency, reproducibility, sensitivity, and compatibility and investigate if the bead technology would improve plasma protein profiling in proteomic analysis compared with the depletion method.

 2008 American Chemical Society

Plasma Samples. Human plasma was provided from one healthy donor at AstraZeneca R&D, Mo¨lndal. The plasma sample was centrifuged at 2000 rpm for 10 min prior to splitting into aliquots and stored at -20 °C pending biochemical analysis. The study was conducted in accordance with AstraZeneca’s ethical guidelines. Bead Treatment of Plasma Samples Using One-Step Elution. The ProteoMiner kit (BioRad, Hercules, CA, USA) for 2-D gel analysis was used according to the manufacturer’s instrucJournal of Proteome Research 2008, 7, 4191–4198 4191 Published on Web 08/09/2008

technical notes

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Table 1. Plasma Protein Recovery of Bead Treated Plasma Samplesa fraction

plasma protein recovery after bead treatment (%)

CV (%)

Flow through Elution Total

84.5 2.3 86.8

7.6 9.1 6.7

a Recoveries were calculated from quantification of total protein concentrations. All values are based on six independent bead experiments. The recovery of the crude plasma sample in the flow through fraction was calculated from a measured protein concentration in plasma (78 g/L).

Figure 1. SDS-PAGE analysis of plasma before and after bead treatment using one-step elution from six independent bead experiments. Six × 1 mL of human plasma were loaded on six bead columns and eluted by a buffer containing urea, CHAPS, and acetic acid. The proteins were separated by 4-12% NuPAGE Bis-Tris gels using MES buffer and stained with SYPRO Ruby. From left to right,1–9 normal human plasma, flow through, bead eluate replicates 1-6.

tions. The plasma sample was analyzed in six independent bead experiments. Briefly, six spin columns were washed with water 2 × 5 min, and then by wash buffer 2 × 5 min, and thereafter, 1 mL of the plasma was applied to each of the columns for incubation during 2 h. The columns were then washed with wash buffer for 3 × 5 min. After all buffers have been removed, 100 µL of rehydrated elution reagent (buffer containing urea, CHAPS, acetic acid) was added and incubated for 3 × 15 min. All three elutions were pooled and collected from the six independent bead experiments. Bead Treatment of Plasma Samples Using Four Steps Elution. The ProteoMiner kit (BioRad, Hercules, CA) for sequential elution was used according to the manufacturer’s instructions. The plasma sample was analyzed in two independent bead experiments. Briefly, the washing and incubation steps of plasma samples were identical as in the ProteoMiner kit for 2-D gel analysis. However, the proteins were eluted in a four-step procedure, including elution reagent 1 (1 M NaCl, 20 mM HEPES, pH 7.5), elution reagent 2 (200 mM glycin, pH 2.4), elution reagent 3 (60% ethylene glycol), and elution reagent 4 (33.3% isopropanol, 16.7% acetonitrile (ACN), 0.1% trifluoro acetic acid (TFA)) at each elution step for 2 × 10 min. The two elutions from reagent 1-4 respectively were collected and pooled. Depletion. For comparison with the bead technology, one depletion column, Multiple Affinity Removal column, MARS14 (Agilent Technologies, San Diego, CA) have been used for the removal of the 14 most abundance proteins in plasma 4192

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samples. The depletion procedure was performed at room temperature according to manufacturer’s instructions. The plasma sample was analyzed in two independent depletion experiments. The LC system used for chromatographic depletion columns consisted of a binary Gynkotek P580 HPLC pump (Gynkotek, Munchen, Germany), a Gilson Aspec XL autosampler (Gilson, Villies-le-Bel, France), and a Gynkotek UV 340U detector (Gynkotek, Munchen, Germany). Data acquisition as well as gradient control was performed with the Chromeleon software, version 6.0 (Dionex, Sunnyvale, CA). Protein Assays. Total protein content was determined using the RC-DC protein assay (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions with bovine serum albumin as standards (Pierce, Rockford, IL). SDS-PAGE analysis was done by the NuPAGE system (Novex precast gels, San Diego, CA, USA) using 4-12% Bis-Tris gels (10 wells) and the NuPAGE (2-(N-morpholino)ethane sulfonic acid) MES buffer system (1 M MES, 1 M tris, 69 mM SDS, 20 mM EDTA) as running buffer. The gels were stained with SyproRuby (Molecular Probes, Eugene, OR, USA. SELDI-TOF-MS. For profiling of bead treated, depleted and crude plasma samples (corresponding to a total concentration of 1 µg), cationic (CM10) ProteinChip arrays and 50 mM ammonium acetate, pH 6 were used. The ProteinChip arrays were prepared using a BioMek 2000 Laboratory Automated Workstation (Beckman Coulter). The samples were incubated for 30 min and the spots were then washed 3 × 5 min with 150 µL buffer to remove unspecific protein binding and salts. The ProteinChip arrays were also washed two times with deionized water. Sinapinic acid (SPA, Sigma-Aldrich, St. Louis, MO) was used as matrix. A saturated solution of SPA, (12.5 mg/ mL), diluted 1:2 v/v in 50% ACN and 0.5% TFA, were applied twice (1 µL) to each dried sample spots to form crystals. ProteinChip arrays were analyzed in PBS II ProteinChip Reader (BioRad). Desorption/ionization was generated using an average of 220 laser shots at two different laser intensities, one at 190-200 for peptides/ proteins in the 2-10 kDa range and one at 200-210 for proteins in the 10-80 kDa range. The ionized molecules were detected and their molecular masses were determined according to their time-of-flight. Time-offlight mass spectra were collected in positive ion mode. External calibration of the ProteinChip Reader was performed using the all-in protein/peptide standard (Biorad) diluted in SPA matrix. The spectra were analyzed using Ciphergen Express Client version 3.0.6. Protein profile comparisons were performed after baseline subtraction and normalization on total ion current of all spectra included in the same experiment. 2-D DIGE. A volume corresponding to 50 µg protein (4-7 µL of the bead treated plasma samples) was diluted in DIGE sample buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris, pH 8.5) to 15 µL. To ensure that the pH was between 8.0-9.0, 2 µL of 1 M Tris solution was added. The bead treated plasma samples (n ) 6) were labeled with CyDye DIGE Fluors (Amersham, GE Healthcare) prior to 2-D gel analysis. The reconstitution of CyeDyes was done according to the manufacturer’s instruction. One internal standard was done of mixing one part of all six bead treated plasma samples together. CyeDye DIGE Fluors (CyeDye 2, 3 or 5) were then added to the protein lysate so that 50 µg of proteins is labeled with 400 pmol of stain dye. The reaction was done on ice in the dark for 30 min. Adding 10 µL of 10 mM of lysine stopped the labeling reaction. Samples (the internal standard labeled with CyeDye 2 and two of the bead treated plasma samples labeled with CyDye 3 and CyeDye

Combination of Beads with SELDI-TOF-MS and 2-D DIGE

technical notes

Figure 2. SELDI-TOF-MS analysis of plasma before and after bead treatment using one elution step from six independent bead experiments compared with depleted plasma. Six × 1 mL of human plasma were loaded on the bead columns and eluted by a buffer containing urea, CHAPS, and acetic acid. The proteins were analyzed on CM10 protein chip arrays using ammonium acetate buffer, pH 6.5. Table 2. Plasma Protein Recovery of Sequential Bead Treated Plasma Samplesa Fraction

Plasma protein recovery after bead treatment (%)

Flow through Elution 1 Elution 2 Elution 3 Elution 4 Elution 1-4 Total

76.9 0.46 1.05 0.64 0.28 2.43 79.4

CV (%)

9.4 15 16 7.0 3.0 9.4

a

Recoveries were calculated from quantification of total protein concentrations. All values are based on two independent bead experiments. The recovery of the crude plasma sample in the flow through fraction was calculated from a measured protein concentration in plasma (78 g/L).

5) were mixed and an equal volume of 2× sample buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris, 20 mM DTT, 0.05% IPG buffer, pH 8.5) was added. Depleted plasma samples (n ) 2) from the MARS 14 column were precipitated by the UPPA kit (G-Biosciences, St Louis, MO). The air-dried pellets corresponding to 50 µg were dissolved in 10 µL of DIGE buffer and the DIGE labeling procedure was performed as described above. Immobiline DryStrips (pH 3-11 nonlinear, 24 cm, Amersham Biosciences) were used for iso electric focusing (IEF). Each strip

Figure 3. SDS-PAGE analysis of plasma before and after bead treatment using 4-step elution from two independent bead experiments. Two × 1 mL of human plasma were loaded on two bead columns and eluted by four buffers using (1) NaCl and HEPES, (2) 60% ethylene glycol, (3) glycine buffer, and (4) 33.3% IPA, 16.7% ACN, 0.1% TFA. The proteins were separated by 4-12% NuPAGE Bis-Tris gels using MES buffer and stained with SYPRO Ruby. From left to right,1–9 normal human plasma, flow through, bead eluates 1-4 from two replicates.

was first rehydrated for 12 h in the Destreak solution and the protein sample was added using cup loading. IEF was carried out in an IPGphor unit (Amersham Biosciences) with the following focusing program: 150 V in 2 h, 300 V for 3 h, 600 V Journal of Proteome Research • Vol. 7, No. 9, 2008 4193

technical notes

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Figure 4. SELDI-TOF-MS analysis of plasma before and after bead treatment in two independent bead experiments using four elution steps on CM10 protein chip arrays using ammonium acetate buffer, pH 6.5.

Figure 5. Representative 2-D profiles for (a) bead treated plasma compared with (b) depleted plasma using the MARS14 column from Agilent. Using beads clearly improved the resolution and increased the intensity of the low abundance proteins compared with 2-D analysis of depleted plasma samples.

for 3 h, 2000 V for 3 h, 2000-8000 V for 3 h, and 8000 V for 3 h. Maximum 50 µA was applied per gel strip. Before the strips were applied to the second dimension, they were equilibrated in two steps, first 15 min in 65 mM DTT and then 15 min in 260 mM iodacetamide for reducing and alkylating the cysteins in the proteins. Both solutions also contained 6 M urea, 30% glycerol 2% SDS, and 50 mM TrisHCl pH 8.8 and 0.007% bromophenol blue. The strips were attached to the top of the 12.5% precast Optigel-LF Pro TrisGlycine SDS-PAGE gels (Optigel, Nextgensciences) using 0.6% agarose (Cambrex BioSciences, Rockland, ME) dissolved in running buffer. Gels were mounted in a Protean Plus Dodeca cell (Bio-Rad) filled with Tris/Glycine/SDS running buffer (BioRad, 24 mM Tris Base, 192 mM glycine and 0.1% SDS) buffer. Electrophoresis was conducted with continuous mixing and cooling at 60 mA/gel for one hour and 90 mA/gel until the tracking dye, bromophenol blue, had reached the anodic side of the gel, approximately seven hours, maximum 500 V and 500 W, 24 °C. 4194

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Progenesis SameSpots (version 7.1.0) (Nonlinear Dynamics, Ltd.) was used for image analysis. The program was used for spot detection, quantification and matching of the gels. Identification of Proteins from 2-D Gels Using Mass Spectrometry. Selected spots were excised from the 2-D gel for identification with MS and MS/MS. Punch-outs were placed into a 96 well Thermo-Fast tube plate (Advanced Biotechnology, Ltd., UK) and destained two times in 70% ACN, 25 mM NH4HCO3. The gel pieces were then dried, and then 0.1 µg trypsin in 25 mM NH4HC03 (Promega, Madison, WI, USA) was added to each gel piece and digestion took place overnight at 37 °C. The peptides were extracted from the gel pieces by the addition of 15 µL extraction solution (1% ACN, 1% formic acid) and incubated for one hour. Identification of proteins were performed with MALDI TOF/ TOF or with nano-LC LTQ-Orbitrap. For MALDI TOF/TOF analysis, 0.7 µL, two times, of the samples were manually applied to a 192-position target plate and allowed to dry prior to application of 0.5 µL matrix (R-cyano-4-OH-cinnamic acid,

technical notes

Combination of Beads with SELDI-TOF-MS and 2-D DIGE

Figure 6. Identification of proteins after the bead treatment. Using the bead technology highly improved the spot resolution and resulted in an increased possibility of identification of lower abundance proteins.

diluted 1:1 with 50% ACN, 0.1% TFA, 10 mM ammoniumphosphate). Selected samples were concentrated on an in-house prepared Stage tipdisc tip C18 column13 and eluted with 1 µL 70% ACN, 0.1% TFA directly on the steel target plate for analysis on a 4700 Proteomics Analyzer MALDI TOF/TOF mass spectrometer (Applied Biosystems). Internal calibration was performed by using two trypsin auto digestion fragments (Mr ) 842.51 and 2211.10 Da) Nano-LC-MS/MS analysis was performed on a 20 cm × 50 µm i.d. fused silica column packed in-house with ReproSil-Pur C18-AQ 3 µm porous particles, connected to an LTQ-Orbitrap mass spectrometer (ThermoFisher). Two to 4 µL sample injections were made (Agilent 1100 autosampler) depending on the spot intensity and the peptides were trapped on a precolumn, 4.5 cm × 100 µm i.d., before separation. After 5 min linear run with 0.1% formic acid, the gradient was 10-50% ACN during 5-30 min (Agilent 1100 binary pump), the flow rate was 200 nL/min after a T splitter, and the eluent was electrosprayed from the emitter tip. The instrument was operated in data-dependent mode to switch between Orbitrap (FT-MS) survey scan and ion trap (IT-MS/MS) of the three most abundant doubly or triply protonated ions. Database searching was performed by Mascot peptide mass fingerprinting using the GPS Explorer Software (Applied Biosystems) (s/n ratio 15), MASCOT Daemon database search software session 2.2.0 (Matrix Science) and Mascot MS/MS ion searchsoftware’s(MatrixSciences,London,UK).ForLC-ESI-MS/ MS data, the peak list was generated with extract_msn.exe included in the ThermoElectron Bioworks browser version 3.3, using data import filter for mass 400-3000, minimum number of peaks 5, threshold 2000 and the precursor charge state was calculated by XCalibur version 2.0 SR2. The search method used database information from inhouse PDB, PIR, SwissProt, and Trembl databases using mouse/ rat/human sequences. Variable modifications were set to oxidation on methionine, mass tolerance for MALDI-MS and MS/MS analysis of (35 ppm and a fragment mass tolerance

of (0.25 Da and a mass tolerance for LTQ-Orbitrap MS/MS analysis of (5 ppm and a fragment mass tolerance of (0.8 Da were used. The criterium for identity was fulfilled if a significant score in MALDI-MS/MS match in the database and/or two significant separate peptide scores for LC-ESI LTQ-Orbitrap MS/MS data were obtained.

Results and Discussion Evaluation of the bead technology, using one step elution, regarding effiency, reproducibility, sensitivity and compatibility was made through quantification of total protein, 1-D gel electrophoresis, 2-D DIGE, SELDI-TOF-MS using six independent bead experiments. The total protein content was used to investigate the reproducibility and protein recovery of the bead treated plasma samples. The calculated plasma protein recoveries are presented in Table 1. Total protein concentration of untreated plasma was 76 mg/mL. The mean recovery of the eluted bead treated plasma samples was 2.3% (Table 1) compared with 9-10% recovery after the depletion process using the MARS 14 column (based on two independent depletion experiments). The bead treatment showed reproducibility lower than 10% in the determination of total protein content, which is a reasonable error in proteomic analysis. As indicated in Table 1, some proteins, approximately 10%, were left on the column after the elution step. 1-D SDS-PAGE of bead treated plasma showed identical profiles of the six independent bead experiments (Figure 1). Figure 2 demonstrates the corresponding SELDI-TOF-MS profiles of the six replicates of the bead treated plasma samples compared with depleted plasma. Many more peptides and proteins were detected after bead treatment compared with analysis of crude and depleted plasma. The CV across six replicates was found to be 18%, also a reasonable error in a proteomic analysis of biological samples. Evaluation of beads using four step elutions was evaluated through quantification of total protein, 1-D gel electrophoresis, and SELDI-TOF-MS based on two independent bead experiJournal of Proteome Research • Vol. 7, No. 9, 2008 4195

technical notes

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Table 3. MS/MS Identification of Proteins Detected by 2-D DIGE from Bead Treated Plasma Samples database entrya*_HUMAN

4196

protein name

accessionb

ACTA ALBU APOA1 APOA1

Actin, aortic smooth muscle albumin apolipoprotein A1 apolipoprotein A1 (fragments)

P62736 P02768 P02647

APOA2 APOA4

apolipoprotein A2 apolipoprotein A4

P02652 P06727

APOC1 APOC2 APOC3 APOD

apolipoprotein apolipoprotein apolipoprotein apolipoprotein

P02654 P02655 P02656 P05090

APOE

apolipoprotein E

P02649

CERU

ceruloplasmin

P00450

C4BP

C4 binding protein

P04003

CO3

complement factor 3A

P01024

CO4A

complement factor 4A

P0C0L4

CO4B CLUS

complement factor 4B apolipoprotein J

P0C0L5 P10909

C1QB C1QC C1S

complement C1q subcomponent subunit B precursor complement C1q subcomponent subunit C recursor complement factor C1s

P02746 P02747 P09871

DCD FCN2 FCN3 FHR1 FHR5

dermcidin ficolin-2 precursor ficolin-3 precursor complement factor H-related protein 1 precursor complement factor H-related protein 5 precursor

P81605 Q15485 O75636 Q03591 Q9BXR6

FIBA FIBA

fibrinogen alpha fibrinogen alpha (fragments)

P02671

FIBB FIBB

fibrinogen beta fibrinogen beta (fragments)

P02675

FIBG FIBG

fibrinogen gamma fibrinogen gamma (fragments)

P02679

C1 C2 C3 D

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Mascot scorec

no. of peptidesd

MS/MS method

159 300 43 354 211 145 152 1268 114 210 122 92 164 257 240 205 257 1235 1082 1298 1121 1351 367 422 48 702 1179 170 75 365 98 136 75 415 347 399 117 75 345 308 632 688 309 592 263 191 95 114 76 66 132 127 239 46 72 774 798 230 304 274 128 83 74 1360 62 348 171 115 133 161 123 306 144 452 1146 1155 117

4 20 1 12 8 8 4 65 3 6 3 3 2 5 5 4 8 30 26 27 21 32 10 10 2 16 11 7 2 8 3 4 2 8 8 10 3 2 12 9 16 17 10 15 9 6 3 2 1 2 2 3 1 2 4 19 22 4 8 5 3 3 2 33 3 9 4 3 3 3 3 8 5 10 27 26 6

LC-ESI MALDI MALDI LC-ESI LC-ESI LC-ESI LC-ESI LC-ESI MALDI MALDI LC-ESI LC-ESI MALDI LC-ESI LC-ESI LC-ESI LC-ESI LC-ESI LC-ESI LC-ESI LC-ESI LC-ESI LC-ESI LC-ESI MALDI LC-ESI LC-ESI LC-ESI LC--ESI LC-ESI LC-ESI LC-ESI LC-ESI LC-ESI LC-ESI LC-ESI MALDI LC-ESI LC-ESI LC-ESI LC-ESI LC-ESI LC-ESI LC-ESI LC-ESI MALDI LC-ESI MALDI MALDI LC-ESI MALDI MALDI MALDI MALDI MALDI LC-ESI LC-ESI MALDI LC-ESI LC-ESI LC-ESI LC-ESI LC-ESI LC-ESI MALDI LC-ESI LC-ESI MALDI MALDI MALDI MALDI LC-ESI LC-ESI LC-ESI LC-ESI LC-ESI LC-ESI

technical notes

Combination of Beads with SELDI-TOF-MS and 2-D DIGE Table 3. Continued database entrya*_HUMAN

accessionb

protein name

IGHG1

Ig gamma 1 chain C

P01857

ITIH4 KAC

Interalpha-trypsin inhibitor heavy chain H4 Ig kappa chain C region

Q14624 P01834

KV305

Ig kappa chain V-III region WOL

KV106

Ig kappa chain V-I region EU

MASP2 PLMN PON1

mannose-binding lectin serine protease 2 precursor plasminogen paraoxonase 1

O00187 P00747 P27169

PROS SAMP

vitamin K dependent protein S serum amyloid P component precursor

P07225 P02743

THRB TTHY VTNC

prothrombin transthyretin (TTR) vitronectin

P00734 P02766 P04004

ZPI 1433Z 1433G 1433S

serpin 14-3-3 protein gamma 14-3-3 protein zeta/ delta 14-3-3 protein

Q9UK55 P63104 P61981 P31947

Mascot scorec

no. of peptidesd

MS/MS method

1042 368 179 129 187 729 453 376 102 76 150 160 201 192 121 151 121 180 127 143 184 159 73 260 105 181 218 73 116 manual sequencing 114 236 79 372 375 122

22 12 7 2 5 21 16 14 3 3 4 5 3 3 3 3 3 4 3 2 5 4 4 7 3 4 5 2 2 1 3 4 2 11 14 6

LC-ESI LC-ESI LC-ESI LC-ESI LC-ESI LC-ESI LC-ESI LC-ESI MALDI MALDI MALDI MALDI LC-ESI LC-ESI MALDI LC-ESI LC-ESI MALDI LC-ESI LC-ESI LC-ESI MALDI LC-ESI LC-ESI MALDI LC-ESI LC-ESI LC-ESI MALDI MALDI LC-ESI LC-ESI MALDI LC-ESI LC-ESI LC-ESI

a Uniprot knowledgebase entry, all entries with extension _HUMAN. b AC is primary accession number from Uniprot knowledge base. c Proteins were identified by mascot database search of all MS/MS in Uniprot. All matches are identified significantly. Identified proteins are considered as positive match on at least a 95% significance level (p < 0.05) corresponding to a significance threshold ionscore of 28. d Number of tryptically peptides that match the identified protein. At least one matching peptide for each identified protein must fulfill significance criteria.

ments. The total protein recovery of the sequential elution of the beads was in the same range as for the one step elution using the buffer containing urea and CHAPS (Table 2). SDSPAGE analysis shows that the four different elutions from the two plasma replicates were almost identical (Figure 3). Figure 4 demonstrates the corresponding SELDI-TOF-MS profiles of the bead treated plasma samples. The number of peaks detected by SELDI-TOF-MS on CM10 protein chip arrays were highly increased in the sequential bead treated plasma samples (n ) 330) as compared with single elution of the beads using the buffer containing urea and CHAPS (n ) 91), depletion (n ) 58) and by direct analysis of crude plasma samples (n ) 48). However, some overlap between the individual bead fractions were observed both by SELDI-TOF-MS and SDS-PAGE (Figures 3 and 4). Bead treated plasma, using one step elution, was only evaluated by 2-D DIGE, since four fractions from the sequential elutions of the beads will be a limitation regarding throughput. The combination of bead technology and 2-D DIGE is very simple, since the plasma proteins are eluted with a buffer containing urea and CHAPS, which is directly compatible with 2-D gel electrophoresis. The pH of the elution buffer only has to be adjusted prior to the DIGE labeling procedure by adding 2 µL of 1 M Tris buffer. Figure 5 shows representative 2-D DIGE profiles of bead treated plasma compared with depleted plasma. The bead treated plasma exhibits many more spots (n ) 1100) in the entire pH interval as compared with depleted plasma (n ) 675). Bead treatment enabled improved intensity

of low abundance proteins and detection of several new protein spots and peaks. The dynamic range reduction is really evident in the 2-D map of the bead treated plasma compared with depleted plasma. The CV of the combination of bead technology and the 2-D DIGE of plasma samples was 14.2% for 650 spots based on six independent bead experiments, whereas the CV was slightly higher for the combination of beads and SELDITOF-MS. Identification of protein spots from the 2-D gel of the bead treated plasma samples are shown in Figure 6 and the results from MS and MS/MS analyses are reported in Table 3. The dominating proteins after bead treatment of plasma samples have been identified to fibrinogen R, β and γ. Other proteins have been identified, for example different kinds of complement factors, proteins involved in immunological reactions, apolipoproteins, and cytoskeletal proteins in the concentration range of µg/L to g/L in plasma, which shows the ability of the bead technology to detect high, medium and low abundance proteins. Different isoforms of 14-3-3 has for example been identified on the 2-D DIGE gel of bead treated plasma, and this protein is normally present in plasma at a concentration of 50 µg/L.14 Our study provides a comprehensive investigation between the bead technology and the MARS-14 depletion column. Based on SELDI-TOF-MS and 2-D DIGE, it was concluded that the beads offered the most promising prefractionation approach with good efficiency, and reproducibility. Another important Journal of Proteome Research • Vol. 7, No. 9, 2008 4197

technical notes advantage of using bead treatment in combination with 2-D DIGE and SELDI-TOF-MS is the simplicity of the beads, instead of depletion. The eluates from the spin columns can be used immediately, without any processing of the sample, except for adjusting the pH of the eluate from pH 3.5 to 8.5, which is the optimal pH of the DIGE labeling process prior to the 2-D analysis. It is also important for a prefractionation method to generate a low number of fractions; several fractions affected the throughput of the downstream analysis. The flexibility of using different numbers of elution steps in the bead technology, from one step to the sequential four-step procedure, is also an advantage. The four-step elution procedure is not a limitation in combination with SELDI-TOF-MS, but of course in combination with 2-D gel electrophoresis. However overlaps between the elution fractions were obtained, which might support that one-step elution is preferable. Furthermore SELDI-TOF-MS and 2-D DIGE of bead treated plasma demonstrated an increased resolution of low abundance proteins in a reproducible fashion with subsequent identification of new proteins in all molecular weight areas. For proteomic studies involving plasma/serum samples, the bead strategy is attractive because the sample will retain representatives of all proteins within the sample.

Conclusion Our results show that the bead strategy is an efficient and simple prefractionation method and in combination with 2-D DIGE and SELDI-TOF-MS the possibility to reach into the areas of low abundant proteins in studies of the plasma proteome is increased.

Acknowledgment. We are very grateful to Tasso Miliotis for the depletion experiments. References (1) Gianazza, E.; Arnaud, P. Chromatography of plasma proteins on immobilized Cibacron Blue F3-GA. Mechanism of the molecular interaction. J. Biochem. 1982, 203, 637–641. (2) Bjo¨rhall, K.; Miliotis, T.; Davidsson, P. Comparison of different depletion strategies for improved resolution in proteomic analysis of human serum samples. Proteomics 2005, 5 (1), 307–317.

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