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Detection and quantification of lysine acetyl-alteration using antibody microarray Background: Lysine acetylation is a reversible and dynamic post-translational modification on proteins, and plays an important role in diverse biological processes. Technological limitations have so far prevented comparative quantification of lysine acetylation in different samples. Results: We developed a method to efficiently study lysine a­cetylation on individual proteins from complex mixtures, using antibody microarrays to capture individual proteins followed by detection with lysine acetyl antibody. By profiling both protein and acetylation variations in multiple samples using this microarray, we found cancer-associated lysine acetylation alteration on VEGF in the serum of hepatocellular carcinoma patients. Conclusion: Microarrays of lysine acetylation are highly effective for detecting acetylation, and should be useful in identifying and validating disease-associated acetylation alterations as bi­omarkers under both normal and pathological circumstances.

Numerous studies have shown that lysine a­cetylation is one of the most important posttranslational modifications (PTMs) on proteins and regulates key physiological processes such as chromatin remodeling, gene expression, cellular metabolic regulation and protein stability [1–3]. The process of lysine acetylation is an important issue to be addressed. The aberrant lysine acetylation may affect tumor-suppressor genes leading to the occurrence and development of several diseases, such as cancer and neurodegenerative and cardio­vascular disorders [4–6]. Therefore, efficient detection of lysine acetylation on biomarkers in complex biological samples would be useful, including characterization of diseaseassociated acetylation alterations, identification of new diagnostic biomarkers, and therapeutic strategies based on interfering with acetyl­ ation-mediated processes or targeting cancer ­acetylation [6,7]. Although lysine acetylation emerges as a frequently occurring PTM and has important functions, it has relatively low abundance and, thus, requires enrichment over non-acetylated proteins for detection in high-throughput proteomics. Both factors (the weak affinity of acetyl-antibody and no efficient enrichment technique specific for lysine-acetylated proteins) have limited the large-scale research of lysine acetylation [8]. Recently, technologies of MS coupled with stable isotope labeling of amino acids in cell culture, immunoaffinity purification of lysine acetyl peptides and chromatographic separation have been reported, which

have enabled sensitive, accurate and quantitative analysis of thousands of novel lysine acetylation sites [6,9–11]. As we know, MS usually requires prefractionation of samples, which makes operation more complicated and may require substantial amounts of samples, particularly for crude samples such as serum and plasma. So it is a major handicap to study small amounts of clinical specimens by MS [12,13]. Meanwhile, it is time-consuming, laborious and expensive for MS to screen large sample sets [12,13]. Microarray is advantageous when dealing with precious clinical samples [14–16] due to its multiplexing capability (acquisition of many data points in parallel) and miniaturization (small consumption of reagents and samples) without prefractionation. The first generation of microarrays have already been used in comparative proteomics for profiling detailed protein expression and PTMs [17,18] such as glycosylation [19–21] and phosphorylation [22,23], which demonstrated its potential for discoveries within the field of disease proteomics. Chen et al. used the glycan microarray to identify and develop potential biomarkers by profiling glycan changes on particular proteins in various disease states with small amounts of samples and reagents [18]. We have developed a method to detect and quantify lysine acetylation of a targeted protein, based on the use of lysine acetyl antibody to probe acetylation on protein captured by a proteinspecific antibody. We utilized acetylated bovine serum albumin (BSA-Ac) as the standard acetylated protein to explore and establish this method.

10.4155/BIO.13.191 © 2013 Future Science Ltd

Bioanalysis (2013) 5(20), 2469–2480

Feina Yao1,2 , Ying Li1,2 , Pengyuan Yang1,2 , Yinkun Liu3 & Huizhi Fan*1 Department of Chemistry, Fudan University, Shanghai 200433, China 2 Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China 3 Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai 200032, China *Author for correspondence: Tel.: +86 21 5423 7416 Fax: +86 21 5423 7416 E-mail: [email protected] 1

ISSN 1757-6180

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R esearch A rticle | Key Terms Acetylation: Dynamic and reversible post-translational modification process with i­mportant implications in c­ellular regulation.

BSA-Ac: Bovine serum albumin is modified with acetyl groups by chemical derivatization.

VEGF: Plays an important role in angiogenesis.

Hepatocellular carcinoma: One of the most common and malignant tumors in the world.

3D aldehyde protein chip slides: A kind of microarray substrate that can maintain protein activity and is suitable for protein microarray c­onstruction.

SmartArrayer™ 136:

Multipurpose microarray spotter that can print liquid samples onto various substrates and build the protein chip.

Capture antibodies:

Polyclonal antibodies often used as the capture antibodies to pull down the antigen as much as possible.

Detection antibodies:

Monoclonal antibodies often used as the detection antibodies to detect the captured antigen.

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We then applied this microarray to detect the cancer-associated acetylation alteration on VEGF in sera between hepatocellular carcinoma (HCC) and control patients. Detection of protein and acetylation on targeted protein in parallel will help us identify and validate disease-associated acetylation alterations on biomarkers under both normal and pathological circumstances. Experimental „„Chemical & reagents The 3D aldehyde protein chip slides (CapitalBio Corporation, China) were used as micro­ array substrates. LuxScan™10K (CapitalBio Corporation) was used for microarray imaging and data analysis. SmartArrayer™ 136 (CapitalBio Corporation) was used to spot liquid samples onto microarrays. Mili-Q (Millipore, USA) was used to make ultra-pure water. Goat serum (Biosynthesis Biotechnology, China) was used as blocking solution. Streptavidin-cyanine dye 3 (Cy3) (Invitrogen, USA) was used as fluorescent reagent. BSA antibody (B-140, Santa Cruz Biotechnology, CA, USA) was used as the capture antibodies. BSA antibody (F-18, Santa Cruz Biotechnology) and lysine acetyl antibody (PTM BioLab, China) were biotinylated with the Biotin Labeling KitNH2 (Dojindo Laboratories, Japan) as detection antibodies. Human VEGF 165 Biotinylated Affinity Purified PAb (BAF293, R&D, USA) was used as the detection antibody. Human VEGF MAb (MAB293, R&D, USA) was used as the capture antibody. Recombinant human VEGF 165 (293-VE-010, R&D, USA) was used as the standard protein. The BSA-Ac was made from BSA (Bovogen Biologicals Pty, Ltd, Victoria, Australia) following the protocol as described by Guan et al. [10]. 1 × phosphatebuffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2HPO4, 1.4 mM KH2PO4 and pH = 7.4) was used as buffer solution. Biotin IgG was used as the positive control. Tris-buffered saline/Tween® 20 (TBST, 20 mM Tris, 137 mM NaCl, 0.1% Tween 20 and pH = 7.4) was used as washing buffer. Mini-PROTEAN® 3 Cell (Bio-Rad, Hercules, CA, USA), horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) and polyvinylidene difluoride (PVDF) membranes (Millipore) were applied in western blot analysis. All buffer solutions were prepared with ultra-pure water (Millipore). Chemiluminescence (Amersham Biosciences, GE Healthcare Life Sciences) was used for western blot. Bioanalysis (2013) 5(20)

Sera were collected from 46 patients (23 HCC and 23 controls) in ZhongShan Hospital (Shanghai, China) with the informed consent of all subjects. All samples were stored at -80°C. „„Microarray

fabrication SmartArrayer 136 system spotted less than 1 nl of antibodies or protein solution onto the 3D aldehyde protein chip slide, which contained 12 arrays. On each array, biotin-lgG solution was printed on the bottom row as a positive control to normalize data from slide to slide, and BSA as negative control on the top row was used to check the nonspecific reaction, following BSA antibody spotted in four other consecutive rows, each row containing eight spots [23]. After spotting, the slides were kept at 4°C overnight to ensure complete receptor immobilization. We imprinted a wax border around each of the arrays to define hydrophobic boundaries, using a custom-built device. Figure 1 shows the flowchart of this microarray. At first, capture antibody and control proteins were spotted onto the surface of a microarray. Then 5% goat serum/TBST was used as a blocking buffer to avoid nonspecific binding. After that, samples were incubated. BiotinBSA antibody or biotin-lysine acetyl antibody as detection antibody was added to recognize samples. Finally, Strepadrew-Cy3 was added to integrate with biotin groups to obtain the amplified fluorescent signals. „„Synthesis

& evaluation of BSA-Ac by SDS‑PAGE & western blot We synthesized the standard acetylated protein as described by Guan et al. [10]. For SDS-PAGE, a 1 mm thick 12% tris-glycine gel was used. BSA and BSA-Ac were boiled for 3 min in the presence of 60 mM SDS and 200 mM DTT prior to loading. Electrophoresis was performed using a Mini-PROTEAN® 3 Cell with 10 mA per gel at the beginning and with 15mA per gel after all proteins had transferred from the stacking gel into the resolving gel. After electrophoresis, the gel was stained using a general R250 Coomassie Brilliant Blue staining method. The proteins on SDS-PAGE were transferred to PVDF membranes by electrophoresis. Membranes were blocked with 5% milk in TBST for 2 h. After rinsing, membranes were incubated with anti-BSA antibody (B-140) (1 µg/ml) or acetyl antibodies (1 µg/ml) in 5% milk/TBST for 2 h, then washed to remove unbound antibodies, membranes were incubated with horseradish future science group

Detection & quantification of lysine acetyl-alteration using antibody microarray

Streptavidin-Cy3

Acetylated protein

Acetyl group

Capture antibody

Detection antobody

Acetyl antibody specific

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Biotin

Sample

Figure 1. Format of the microarray. Samples are incubated on microarrays. The quantitative levels of protein and acetylation are determined from fluorescence intensities.

peroxidase conjugated secondary antibody at a dilution of 1:5000 in 5% milk/TBST for 1 h. After a final wash, proteins were detected by chemiluminescence visualized by las3000. The whole incubation process was done at room temperature. Meanwhile, the washing process was performed with TBST three times, and each time lasted for 5 min. „„Microarray

preparation & operation The microarray was incubated with blocking buffer (5% goat serum in TBST) for 1.5 h with gentle agitation. After washing, 20 µl of samples were added to each array and incubated for 1.5 h. The arrays were then washed to remove unbound samples and clean the surface. Afterwards, detection antibody was added to each array and incubated for 1.5 h. After washing and drying, Streptavidin-Cy3 (10 µg/ml, 20 µl) was added to each array and incubated for 1 h. Finally, the microarray was washed and then scanned to obtain images and result value by LuxScan™10K. All incubation processes were at room temperature. The washing process was performed with TBST three times and each time lasted 5 min. „„Fluorescence

detection & data analysis All arrays were scanned within an experiment set at a single laser power and detector gain setting. Optimal post-translational modification values were chosen for each probe, avoiding more than 10% saturated pixel per spot. All the fluorescence signals were background corrected and filtered for invalid spots which gave a flag not found by the image analysis software or excluded by an outlier test. Mean values of remaining spots were used for down-stream data future science group

analysis. The curves were set up using Origin Pro 8G. The LOD was defined as the lowest concentration of an analyte that microarray could reliably detect and used to evaluate microarray sensitivity. %CV (SD/mean × 100) was used to measure the data reproducibility. Results & discussion „„SDS-PAGE & western blot identification of BSA-Ac To examine the purity and specificity of BSA-Ac synthesized chemically, SDS-PAGE and western blot have been utilized (Figure 2). BSA-Ac showed higher molecular weight in the SDS-PAGE gel (Figure 2A). Western blot confirmed the specificity of acetyl antibody to BSA-Ac (F­igure 2B). In Figure 2C , western blot with BSA antibody was used to check whether the acetyl groups affected the affinity of BSA antibody to BSA-Ac. The BSA-Ac band demonstrated acetyl groups haven’t affected the specific binding between BSA-Ac and BSA antibody. B A Marker

BSA

BSA-Ac

BSA BSA-Ac

Acetyl antibody 1 µg C

1 µg

BSA-Ac

BSA antibody 1 µg

Figure 2. Evaluating the purity and specificity of acetylated bovine serum albumin. (A) SDS-PAGE of BSA-Ac and BSA. (B) Western blotting of BSA-Ac and BSA with acetyl antibody. (C) Western blotting of BSA-Ac with BSA antibody. BSA: Bovine serum albumin; BSA-Ac: Acetylated BSA.

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„„Evaluation

of the specificity & accuracy of microarrays Microarrays might suffer from nonspecific interactions between antibodies and other components. We carried otu a systematic study to determine potential crossreactivity among: capture antibody and detection antibody; antigen and detection antibody; and capture antibody and antigen [24]. Experiments were performed in triplicate and replicated on different days. The results were listed in Table 1. PBS and 5% goat serum were used to check the background of the micro­array. The lower fluorescence signal meant there was little response on background, and no cross­ reactivity between the capture and detection antibodies. When incubating samples of BSA and BSA-Ac, we found the fluorescence intensity of BSA-Ac was much higher than that of BSA, which meant lysine acetyl antibody detected acetylation on BSA-Ac specifically. The lower signals of myoglobin-Ac and myoglobin implied

BSA antibody could not capture other proteins except BSA. As no crossreactions were observed with other samples, the microarray was available for following experiments. Meanwhile, interfering substances in buffer might affect the accuracy of microarrays. We analyzed the interaction by spiking individual BSA or other acetylated proteins into BSA-Ac on microarrays. Experiments were performed in triplicate and replicated on different days. Table 2 showed that the higher BSA concentrations, the lower fluorescence intensity. This side effect was caused by the competitive reaction to capture antibody between BSA-Ac and BSA. However, when concentration of BSA was below 50µg/ml, the deviation was low enough for detection and quantification of acetylation. Meanwhile, other acetylated proteins in low concentration did not change the fluorescence intensity much with low deviations. The results indicated that the microarray could keep its accuracy and was available for following experiments.

Table 1. Specificity of the microarray was checked with different samples. Number Capture antibody (200 µg/ml)

Sample

Detection antibody (2 µg/ml)

Fluorescence CV (%) intensity (a.u.)

1 2 3 4 5 6

PBS 5% goat serum BSA (3000 µg/ml) BSA-Ac (3000 ng/ml) Myolobin-Ac (3000 µg/ml) Myolobin (3000 µg/ml)

Anti-Ac Anti-Ac Anti-Ac Anti-Ac Anti-Ac Anti-Ac

8.33 6.13 4.32 5000.51 30.26 10.80

BSA BSA BSA BSA BSA BSA

4 6 7 10 3 5

Ac: Acetyl; BSA: Bovine serum albumin; PBS: Phosphate-buffered saline.

Table 2. Accuracy of the microarray was checked with different buffers. Protein

Concentration (µg/ml)

Buffer

Concentration (µg/ml)

Fluorescence intensity (a.u.)

Deviation CV (%) (%)

BSA-Ac BSA-Ac BSA-Ac BSA-Ac BSA-Ac BSA-Ac BSA-Ac BSA-Ac

2 2 2 2 2 2 2 2

0 2 10 20 50 100 2 2

4200.03 4100.21 3900.89 3800.54 3400.43 3000.67 4150.34 4258.31

0.00 -2.38 -7.14 -9.52 -19.05 -28.57 -1.19 1.38

5 8 9 10 11 13 8 9

BSA-Ac

4

2

6280.26

3.75

10

BSA-Ac

5

PBS BSA BSA BSA BSA BSA Ova + MyO OvaAc + MyO-Ac OvaAc + MyO-Ac OvaAc + MyO-Ac

2

7300.56

4.10

9

Ac: Acetyl; BSA: Bovine serum albumin; MyO: Myoglobin; Ova: Ovalbumin; PBS: Phosphate-buffered saline.

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Detection & quantification of lysine acetyl-alteration using antibody microarray

A

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C

Replicate spots

Concentrations increase

Replicate arrays

B

Fluorescence intensity (a.u.)

Capture antibody

6000 5000 4000 3000 2000 1000

0 75

Concentrations increase

100 125

Detection antibody

150 175

Concentrations of capture antibody (µg/ml)

200 225

3.0 2.5 2.0 1.5 1.0 0.5

Concentrations of detection antibody (µg/ml)

Figure 3. Different concentrations of capture and detection antibodies influence fluorescence intensity. (A) Concentrations of capture antibody were 75.00, 100.00, 125.00, 150.00, 175.00, 200.00 and 225.00 µg/ml. (B) Concentrations of detection antibody were 0.50, 1.00, 1.50, 2.00, 2.50 and 3.00 µg/ml. (C) The relationship between fluorescence intensities and antibody concentrations.

concentrations of capture & detection antibodies As the optimal concentrations of capture and detection antibodies were variable between certain antigens, related concentrations were optimized by using crisscross serial-dilution analysis in each group [25]. Here we chose BSA-Ac (3.00 µg/ml) as the antigen. The concentration of BSA antibody (B-140) we purchased was 200µg/ml, and we increased the concentration to 225.00 µg/ml with ultrafiltration. After dilution, seven concentrations of BSA capture antibody were spotted (75.00, 100.00, 125.00, 150.00, 175.00, 200.00 and 225.00 µg/ml) onto each array (Figure 3A). Then, six concentrations (0.50, 1.00, 1.50, 2.00, 2.50 and 3.00 µg/ml) of biotin lysine acetyl detection antibody were added on different arrays (Figure 3B). Both the capture and detection antibodies were diluted with PBS. In Figure 3C, the fluorescence intensity kept enhancing as the concentrations of capture and detection antibodies increased. Based on the relationship between concentration and fluorescence intensity, we chose the concentrations of future science group

Fluorescence intensity (a.u.)

„„Optimized

7500 7000 6500 y = 959.01× + 2217.82 6000 R2 = 0.996 5500 5000 4500 4000 3500 3000 2500 2000 1500 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 Concentrations of BSA-Ac (µg/ml)

Figure 4. Relationship between concentration of acetylated bovine serum albumin and fluorescence intensity detected by bio-acetyl secondary antibody. The measurement concentrations in linear relationship were 0.10, 0.50, 0.75, 1.00, 2.00, 3.00, 4.00, 5.00, 6.00, 8.00 and 10.00 µg/ml. Error bars represent the standard deviation of measurements in three times. BSA-Ac: Acetylated BSA.

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capture antibody at 200 µg/ml and detection antibody at 2.0 µg/ml. „„Detection

& quantification of acetylation on BSA-Ac Here we diluted BSA-Ac gradient with 1 × PBS as samples to incubate on the microarray. The measurement concentrations in this experiment were 0.02, 0.05, 0.10, 0.50, 0.75, 1.00, 2.00, 3.00, 4.00, 5.00, 6.00, 8.00 and 10.00 µg/ml. When the concentration was higher than 0.05 µg/ml, the microarray could reliably detect the signal of microarrays. The correlations between fluorescence intensity and concentration of BSA-Ac were shown in Figure 4. The LOD was 0.05 µg/ml with a linear dynamic range from 0.1 µg/ml to 10 µg/ml (R 2 = 0.996). The experiment took less than 20 h and only small amount of sample (20  µl) was used in each array. It was the first time of introducing the microarray for Table 3. Different dilutions of serum affected the interference of serum. Buffer

Dilution

LOD (µg/ml)

CV (%)

PBS Serum Serum Serum Serum

No No 1:10 1:50 1:100

0.05 2 0.5 0.35 0.15

9 18 12 11 10

PBS: Phosphate-buffered saline.

5500

Fluorescence intensity (a.u.)

5000

y = 389.57× + 1837.80 R2 = 0.986

4500 4000 3500 3000 2500 2000 1500

0

1 2 3 4 5 6 7 8 Concentrations of BSA-Ac (µg/ml)

9

Figure 5. The fitting curves about the relationship between the fluorescence intensity and concentrations of acetylated bovine serum albumin diluted in tenfold serum. The measurement concentrations in linear relationship were 0.75, 1.00, 2.00, 4.00, 6.00 and 8.00 µg/ml. Error bars represent the standard deviation of measurements in three times. BSA-Ac: Acetylated BSA.

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detecting and quantifying protein acetylation level rapidly. Experiments were performed in triplicate and replicated on different days. Serum is a complex system which contains many biomarkers and is used in numerous detection systems of diseases. It also contains different kinds of interfering substances such as fibrin, high-abundance proteins and salts that may shield the targeted protein and, hence, hinder specific binding to detection antibodies. Buffers were made by diluting human serum in PBS and BSA-Ac was spiked in these buffers. In Table 3, the detection LOD of BSA-Ac was high in crude serum and decreased when serum was diluted. These results indicated that the human serum had side effects on microarray and this effect was introduced by, at least in part, serum albumin [26,27]. To reduce the interference effect, 1:10 sample dilution was recommended. To determine whether microarray was suitable in complex sample matrices, various concentrations of BSA-Ac were directly spiked into tenfold serum. Based on the method described above, the measurement concentrations in this experiment were 0.25, 0.50, 0.75, 1.00, 2.00, 4.00, 6.00 and 8.00 µg/ml. When the concentration was higher than 0.50 µg/ml, the signal of microarrays could be reliably detected. The linear range was from 0.75 to 8 µg/ml with the LOD at 0.5 µg/ml (Figure 5). Compared to the detection of BSA-Ac diluted in PBS, the sensitivity decreased a little and this side effect was caused, at least in part, by serum albumin [26,27]. Experiments were performed in triplicate and replicated on different days. Accuracy of the microarray was evaluated by standard addition method. Three concentrations were added ranging from the low end (1.50 µg/ml), the mid point (5.00 µg/ml) to the high end (7.00 µg/ml) of the standard curve. Then, we respectively spiked individual BSA-Ac into serum (Table 4). The fluorescence signals of spiked samples were detected and concentrations were calculated according to the standard curves. The deviation varied from -2.41 to 3.07%. Experiments were performed in triplicate and replicated in different days. The results indicated that this method was appropriate for quantification of acetylation on target proteins. „„Parallel

detection of the altered levels of protein & acetylation Another important application of this method was to measure altered levels of protein and acetylation on targeted protein across different conditions. future science group

Detection & quantification of lysine acetyl-alteration using antibody microarray

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Table 4. The accuracy of microarray were measured by standard addition method. Sample

Buffer

Concentration (µg/ml)

Fluorescence intensity (a.u.)

Detected Deviation concentration (µg/ml) (%)

CV (%)

BSA-Ac BSA-Ac BSA-Ac

Serum (1:10) Serum (1:10) Serum (1:10)

1.5 5.0 7.0

2453.91 3694.52 4704.81

1.52 4.88 7.21

10 8 11

1.31 -2.41 3.07

BSA-Ac: Acetylated bovine serum albumin.

To avoid antibody cross-reactivity, we chose two antibodies with distinct epitope, BSA antibody (B-140) as capture antibody and biotin-BSA antibody (F-18) as detection antibody Efor detecting BSA on BSA-Ac [23]. The optimal concentrations of capture and detection antibody were variable between certain proteins. We tried different concentrations of biotin-BSA antibody shown in Figure 6. Based on linear range of concentrations for lysine acetylbinding profiles on BSA-Ac above, we chose the concentration of biotin BSA secondary antibody at 0.10 µg/ml with similar range from 0.50  to 10.00 µg/ml. Then we used this microarray to detect the varied levels of protein and acetylation in mixtures of BSA and BSA-Ac (Figure 6). Mixtures were incubated on replicate sets of microarrays. As detection antibodies, BSA antibody

measured protein levels on one set of microarrays, and lysine acetyl antibody measured acetyl levels on another sets of microarrays (Figure 7). In Figure 7, this microarray was able to detect and quantify both protein abundance and acetylation of protein by comparing distributions of fluorescence intensity. So this method could be used in comparative proteomics, such as comparing acetylation on targeted proteins in different physiological and pathological conditions. Expanded studies would also detect broader acetyl profiles of biomarkers in disease and control samples, which might help diagnose and treat the related diseases. „„Acetylation

variation on VEGF in cancer patients We applied this method to measure the lysine acetylation variation on biomarkers across

10,000 0.1 µg/ml 0.2 µg/ml 0.4 µg/ml 0.6 µg/ml

9000

Fluorescence intensity (a.u.)

8000 7000 6000 5000 4000 3000 2000 1000 0 0

1

2

3

4

5

6

7

8

9

10

11

Concentration of BSA-Ac (µg/ml)

Figure 6. Fluorescence intensities of different concentrations of acetylated bovine serum albumin were detected with different concentrations of biotin-bovine serum albumin antibody shown in four lines. The concentrations of BSA-Ac were 0.50, 1.00, 2.00, 4.00, 6.00, 8.00 and 10.00 µg/ml. The concentrations of biotin-bovine serum albumin antibody were 0.10, 0.20, 0.40 and 0.60 µg/ml. BSA-Ac: Acetylated bovine serum albumin.

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A Protein detection Sample:

BSA BSA-Ac + (1 µg/ml) (2 µg/ml)

B

Acetylation detection

BSA BSA-Ac + (1 µg/ml) (2 µg/ml)

BSA BSA-Ac + (2 µg/ml) (1 µg/ml)

BSA BSA-Ac + (2 µg/ml) (1 µg/ml)

Positive control

Detection:

Biotin-BSA antibody

Biotin-acetyl antibody

Figure 7. A mixture of acetylated bovine serum albumin and bovine serum albumin were incubated on each pair of arrays. The arrays were detected using (A) BSA antibody to detect BSA or (B) acetyl antibody detecting acetylation. BSA: Bovine serum albumin; BSA-Ac: Acetylated BSA.

9000 8000

y = 388.56× - 25.67 R2 = 0.989

Fluorescence intensity (a.u.)

7000 6000 5000 4000 3000 2000 1000 0 0

2

4

6

8

10

12

14

16

18

20

22

Concentration of VEGF (ng/ml)

Figure 8. The fitting curves about the relationship between the fluorescence intensity and concentrations of VEGF diluted in tenfold serum. The measurement concentrations in linear relationship were 0.15, 0.31, 0.63, 1.25, 2.5, 5.0, 10.0 and 20.0 ng/ml. The error bars represent the standard deviation of measurements in three times.

conditions. VEGF plays an important role in regulating angiogenesis and vascular permeability. An aberrantly enhanced level of VEGF is associated with increased tumor growth and metastasis, 2476

Bioanalysis (2013) 5(20)

leading to the spread of solid malignancies including HCC [28]. Serum VEGF is not only a useful prognostic marker in patients with HCC, but also an effective molecular target for antiangiogenic future science group

Detection & quantification of lysine acetyl-alteration using antibody microarray therapy [29,30]. In addition, Pillai et al. found that acetyl­ation played an important role in the angiogenic process [31]. We used parallel protein and lysine-acetyl detection microarrays to look at the variation in the protein and acetylation levels of VEGF over multiple samples, respectively. Poon et al. detected that the median level of VEGF was 0.245 ng/ml with the range from 0.0126 to 1.824 ng/ml in the serum of patients with HCC, and 0.18 ng/ml with the range from 0.0408 to 0.671 ng/ml in healthy controls (p = 0.042) [32]. As the level of VEGF was low in serum, we increased the concentration of capture antibody to 750 µg/ml, and increased the detection linear range from 0.15 to 20 ng/ml with the LOD at 0.05 ng/ml. The detection sensitivity was close to the research by Sachdeva et al. (Figure 8) [33]. Due to the limited concentration of VEGF in serum, we used the VEGF and lysine-acetyl

A

Sample:

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detection antibodies to detect their respective targets in parallel. Incubating a cohort of serum samples (23 from both HCC and control patients) on replicate sets of arrays, the VEGF detection antibody measured protein levels on one set of arrays, while the lysine-acetyl detection antibody measured acetylation levels on another set of arrays (Figure 9). Certain samples showed higher protein and acetylation levels of VEGF in cancer patient sera. Comparing the distributions of acetylation and protein levels indicated some statistical differences (p 

Detection and quantification of lysine acetyl-alteration using antibody microarray.

Lysine acetylation is a reversible and dynamic post-translational modification on proteins, and plays an important role in diverse biological processe...
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