Peptide Profiling with Dynabeads®
A new approach to biomarker discovery and clinical proteomics.
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Serum Peptide Profiling
"Dynabeads® RPC 18 allow for efficient biofluid peptide capture and yield rich serum peptide mass profiles in MALDI-TOF-MS with good batch-to-batch reproducibility of ion intensities"
- Dr. Connie Jimenez, Head, OncoProteomics Laboratory, VUmc Cancer Center Amsterdam, VU University Medical Center, Amsterdam, The Netherlands. |
What is Peptide Profiling?
Discovery and validation of protein and peptide biomarkers in clinical proteomics requires large numbers of patient samples. Traditional methods are complicated, time-consuming and lack standardization.Peptide profiling with magnetic chromatography beads offers quick, reproducible screening and can be automated to run 96 samples in a day from start to finish. The method is reproducible and can be standardized, facilitating clinical and inter-laboratory collaborations.
The process is simple and you can easily capture proteins and peptides from complex samples such as serum, plasma and cerebrospinal fluid (CSF).
Once you have screened the sample you can use columns to perform an in-depth screening. Not only will you conserve your precious samples, you can save months of valuable research time.
The process is simple and you can easily capture proteins and peptides from complex samples such as serum, plasma and cerebrospinal fluid (CSF).
- Incubate your sample with the Dynabeads® of choice
- Use magnetic separation to quickly draw your target peptides to the tube/well wall
- Wash, then elute peptides off the beads
- Perform mass spectrometry and analyse the results.
Once you have screened the sample you can use columns to perform an in-depth screening. Not only will you conserve your precious samples, you can save months of valuable research time.
Table 1 - Dynabeads® vs Conventional Techniques
| Dynabeads® | Conventional Techniques e.g LCMS |
|---|---|
| Reproducible results between experiments - low lot-to-lot variation | High inter-experimental variation |
| Possible to handle 96 samples in a day (from sample prep to data analysis) | 1 day = 1 sample |
| Only need small sample volumes e.g 6 µl serum | Possible to lose sample in column. Clogging causes failed experiments |
| No clogging of columns means no loss of sample or failed experiments | Need to regenerate columns |
| Low cost method | |
Multiple Surface Screening
Multiple Surface Screening of serum samples increases the number of ion peaks observed in a sample. Serum peptide profiling was performed with Dynabeads® RPC 18, Dynabeads® SAX and Dynabeads® SCX using a single serum sample. Aliquots of eluted peptides were analysed by MALDI-TOF mass spectrometry and typical spectra were produced. Fig. 1 shows that distinct spectra are obtained from a single serum sample for each bead type.
Peak lists were compared and the Venn diagram shows percentages of the total ion count for all samples, see Fig. 2.
By using different combinations of Dynabeads® you widen the range of visible ion peaks, increasing the certainty of finding the target peptides that you are searching for.
Peak lists were compared and the Venn diagram shows percentages of the total ion count for all samples, see Fig. 2.
By using different combinations of Dynabeads® you widen the range of visible ion peaks, increasing the certainty of finding the target peptides that you are searching for.
 Figure 1. Distinct spectra are obtained for each bead type from a single sample. |
 Figure 2. Percentages of ion counts from peak lists |
Table 2 - Reproducibility between lots gives results you can trust
| Dynabeads® RPC 18 lot number | Average number of ion peaks* (N=40) | STD DEV | % CV |
|---|---|---|---|
| G 50810 | 280 | 32 | 11 |
| G 72000 | 288 | 26 | 9 |
| H 31200 | 289 | 28 | 10 |
| All lots (N=120) | 286 | 29 | 10 |
| Between lots | 286 | 5 | 1.74 |
Reproducible Results - Use in combination or alone
Reproducibility You Can Trust
Each of the Dynabeads® magnetic chromatography products will give you reproducible spectra because the variation between lots is very low. This table shows the variation in ion peak number per spectrum for serum profiling using three different lots of Dynabeads® RPC 18. In total 40 analyses were performed for each lot using a single serum sample. Aliquots of eluted peptides were analysed by MALDI-TOF mass spectrometry.
As clinical trials/studies take time to complete, using a reproducible, reliable method such as this allows for inter-laboratory studies.
Each of the Dynabeads® magnetic chromatography products will give you reproducible spectra because the variation between lots is very low. This table shows the variation in ion peak number per spectrum for serum profiling using three different lots of Dynabeads® RPC 18. In total 40 analyses were performed for each lot using a single serum sample. Aliquots of eluted peptides were analysed by MALDI-TOF mass spectrometry.
As clinical trials/studies take time to complete, using a reproducible, reliable method such as this allows for inter-laboratory studies.
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Figure 3. Consistent beads give consistent results
Serum peptide profiling was done using Dynabeads® RPC 18 and an alternative method. 80 analyses were performed for each product using a single serum sample. Aliquots of eluted peptides were analysed by MALDI-TOF mass spectrometry, followed by Principal Component Analysis of normalized profiles Fig. 3. Less analytical variation is seen with Dynabeads® RPC 18. |
Published Papers
Several research groups have already developed protocols for other commonly used automated platforms for high-throughput serum profiling. They have found they still get the reproducible results they need, and have saved several days of valuable research time.
To request a copy of the papers, please click here and we can send you a reprint.
Villanueva et al (1) developed an automated protocol to enrich serum peptides from blood that was used as a potential predictive or diagnostic tool. Combining Dynabeads® RPC 18 with their Tecan® Genesis platform, they could simultaneously prepare large numbers of peptides for automated matrix-assisted laser desorption/ionization-TOF MS analysis. Using their sensitive, reproducible method they were able to profile 96
samples in less than 27 hours. Request a copy - click here.
Jimenez et al (2) wanted to establish multi-centre studies of serum profiling and implemented a serum profile processing method using Dynabeads® RPC 18 on a Kingfisher® 96 platform. They found it to be reproducible and robust and they could detect 200 peptides at mass-to-charge ratio (m/z) 800-4000 in each spectrum from serum that had been allowed to clot for 1 hour. Their method can be used by anyone with a MALDI-TOF instrument. Using the Kingfisher 96 took 20 minutes to process 96 samples in parallel, making this an effective way to perform large-scale disease profiling studies. Request a copy - click here.
To request a copy of the papers, please click here and we can send you a reprint.
Villanueva et al (1) developed an automated protocol to enrich serum peptides from blood that was used as a potential predictive or diagnostic tool. Combining Dynabeads® RPC 18 with their Tecan® Genesis platform, they could simultaneously prepare large numbers of peptides for automated matrix-assisted laser desorption/ionization-TOF MS analysis. Using their sensitive, reproducible method they were able to profile 96
samples in less than 27 hours. Request a copy - click here.
Jimenez et al (2) wanted to establish multi-centre studies of serum profiling and implemented a serum profile processing method using Dynabeads® RPC 18 on a Kingfisher® 96 platform. They found it to be reproducible and robust and they could detect 200 peptides at mass-to-charge ratio (m/z) 800-4000 in each spectrum from serum that had been allowed to clot for 1 hour. Their method can be used by anyone with a MALDI-TOF instrument. Using the Kingfisher 96 took 20 minutes to process 96 samples in parallel, making this an effective way to perform large-scale disease profiling studies. Request a copy - click here.
- J Villanueva, K Lawlor, R Toledo-Crow and P Tempst (2006) Automated serum peptide profiling. FNature Protocols, Vol 1, No. 2. 880-891.
- CR Jimenez, ZE Filali, JC. Knol, K Hoekman, FAE Kruyt, G Giaccone, AB Smit and KW Li (2007) Automated serum peptide profiling using novel magnetic C18 beads off-line coupled to MALDI-TOF-MS. Proteomics Clin. Applications 1 (6) : 598 - 604
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