Magnetic particles as powerful purification tool for high sensitive mass spectrometric screening procedures

Authors


Abstract

The effective isolation and purification of proteins from biological fluids is the most crucial step for a successful protein analysis when only minute amounts are available. While conventional purification methods such as dialysis, ultrafiltration or protein precipitation often lead to a marked loss of protein, SPE with small-sized particles is a powerful alternative. The implementation of particles with superparamagnetic cores facilitates the handling of those particles and allows the application of particles in the nanometer to low micrometer range. Due to the small diameters, magnetic particles are advantageous for increasing sensitivity when using subsequent MS analysis or gel electrophoresis. In the last years, different types of magnetic particles were developed for specific protein purification purposes followed by analysis or screening procedures using MS or SDS gel electrophoresis. In this review, the use of magnetic particles for different applications, such as, the extraction and analysis of DNA/RNA, peptides and proteins, is described.

1 Introduction

The sensitive detection of peptides and proteins is one of the greatest challenges in biological sciences and is normally performed by MS or gel electrophoresis. For a full exploitation of the sensitivity of these systems, which is in the range of 10−15–10−12 molar, substances from biological samples must be purified and concentrated efficiently prior to their analysis. For that purpose, different purification methods, such as dialysis using a variety of dialysis membranes, ultrafiltration with molecular cut-off membranes or protein precipitation with trichloroacetic acid or phenol ether are commonly used. Due to unspecific protein binding to the wall of the receptacles or filtration membranes, a considerable to total loss of protein occurs, when working with very low protein concentrations (ng/mL to low μg/mL range).

A powerful alternative compared with the previously described methods is the off-line SPE with small chromatographic particles 1, 2. Using such particles combined with a magnetic core, the purification and concentration of protein samples can be easily performed using magnetic devices. Compared with chromatographic in-line procedures with separation columns, the off-line extraction with magnetic particles offers the possibility of using particles with smaller diameters (0.1–3 μm), resulting in higher binding surface areas. Therefore, these particles should be most suitable for the purification of low concentrated or volume restricted protein solutions.

The high binding surface area, which can be achieved with magnetic particles compared with protein capture on planar chips, also explains the advantage of performing particle-based MS readouts. Chip-based MS approaches are normally realized using chip spots with diameters of 5 mm and sample volumes of 10 μl. A planar chip spot with a diameter of 5 mm results in a total binding surface area of 19.6 mm2. In contrast, when microspheres with a diameter of 1 μm are used for the capture of substances in a sample volume of 10 μl, an overall surface area of 60 000 mm2 can be theoretically obtained. Therefore, a 3000-fold higher binding surface area can be achieved for the binding of substances as compared with planar chips.

Since the first mention of the potential when using magnetic carriers in biological sciences 3, various magnetic purification procedures of biological substances in conjunction with subsequent SDS gel electrophoresis and/or MS analysis have been developed. The most important types of magnetic particle-based applications will be discussed in Sections 2.1–2.8.

2 Magnetic particle-based applications

2.1 Magnetic particle-based isolation and analysis of biotinylated DNA/RNA

The first implementation of magnetic particles in combination with MS analysis of DNA or RNA was published in 1995 by Tang et al.4. In this affinity approach, biotinylated DNA was used as primer for hybridization experiments followed by capturing of the resulting DNA products using streptavidin-coated magnetic particles. Finally, bound DNA products were released from the particles by the addition of matrix and transferred to a MALDI plate, followed by MALDI-TOF-MS analysis. Several other publications dealing with biotinylated DNA/RNA and MS analysis have been published in the past 5–21.

2.2 Magnetic particle-based isolation and analysis of biotinylated peptides and proteins

A similar approach using the streptavidin–biotin technology was performed with biotinylated peptides. At 1996, in a report of the group of G. Bolbach, biotinylated peptides were bound to streptavidin particles and analyzed by MALDI-TOF-MS after the elution with matrix solution 22. The principle of the capture of biotin-tagged peptides with streptavidin-coated particles combined with subsequent MS analysis was also performed with biotinylated proteins or whole cells 23, 24.

2.3 Magnetic particle-based isolation and analysis of tagged recombinant proteins

For the mild purification and subsequent analysis of recombinant proteins, tags other than biotin, e.g. FLAG or 6×His, are normally used. The tagged proteins were isolated and purified with magnetic particles coupled with FLAG-antibodies 25 or Ni2+-nitrilotriacetic acid 26. While recombinant proteins can be received almost in pure forms with these high affinity protein tags, the efficient purification and subsequent analysis of proteins from human body fluids is only possible when suitable antibodies are available.

2.4 Affinity purification of proteins using antibody-coated magnetic particles

For the isolation and MS characterization of low concentrated serum proteins, antibodies coupled to magnetic particles were introduced in 1999 and 2001 by Peter et al. For this purpose, antibodies against prostate specific antigen (PSA) were coupled to magnetic streptavidin-coated polystyrene particles. These “antibody-particles” were used for the isolation of PSA from plasma samples originating from carcinoma patients. The release of PSA was either performed with a digoxigenin solution at neutral pH 27 or under acid conditions 28. The isolated samples were then analyzed by direct MS investigation of the protein or SDS gel electrophoresis followed by in-gel digests and MS investigation of the tryptic peptides. In general, many affinity purification methods described for column chromatography have been also realized on magnetic supports in the last years. An overview has been published by Safarik et al.29 and Horák et al.30.

2.5 Magnetic particle-based isolation and analysis of phosphorylated peptides and proteins

Magnetic particles are also increasingly being used for the specific isolation of phosphorylated peptides and proteins. Since the first attempts of using Fe3+- or Ga3+-loaded sepharose particles for the specific capture of phosphorylated peptides or proteins combined with MS analysis, as described by the groups of Tempst 31 and Tomer 32, novel magnetic microspheres either loaded with or comprised of Fe3+, Ni2+, Ga3+, Zr4+, TiO2, Al2O3 or Ga2O3 were developed for these purposes 33–41.

2.6 Isolation and analysis of glycosylated peptides and proteins using magnetic particles

Another class of magnetic particles was developed for the specific isolation of glycosylated peptides and proteins. For this purpose, magnetic particles were coupled with different lectins, e.g. Con A, weat germ agglutinin, or lens culinaris agglutinin 42–44, or oxidized glycoproteins were bound covalently to hydrazide-modified magnetic silicaparticles 45.

2.7 Magnetic particle-based procedures for MS sera analysis

In contrast to affinity-based applications, magnetic particles can be also used as carriers for the isolation and simultaneous detection of multiple peptides and proteins. Magnetic particles with weak and strong anionic (WAX, SAX), weak and strong cationic (WSX, SCX), hydrophobic (C3, C8 and C18) and other specialized surfaces are available. In 2004, magnetic particles with hydrophobic characteristics were introduced by Villanueva et al. for the MS profiling of serum samples 46. Due to the physically high binding surface area, which can be achieved with these particles, the group of Tempst stated that more sensitive MS readouts can be performed compared with planar chip approaches 46.

Several research papers have been published to date for the MS screening of serum samples using magnetic particles mostly modified with hydrophobic C8 surfaces. Using this type of particles for the generation of MS peptide signatures, the group of Tempst was able to differentiate between healthy volunteers and patients with prostate, breast and bladder cancer after hierarchical clustering and principal component analysis of the peptide ion signals 47. In another study, Villanueva et al.48 also showed that it is possible to differentiate between healthy volunteers and patients with metastatic thyroid carcinoma (tumor sizes of grade four using the TNM classification [http://www.doctorslounge.com/oncology/tnm/index.htm] 49). Moreover, the group of Tollenaar 50 reported that they are also able to distinguish between patients at all stages of breast cancer, including ductal carcinoma in situ, when performing MS peptide screenings using serum and hydrophobic C8 particles. The group of Madsen 51 showed that the peptide isolation from serum samples using magnetic Cu2+-particles combined with a subsequent MALDI-TOF MS readout is suitable even for the early diagnosis of cancer diseases. A review concerning the implementation of magnetic particles with RP or ion exchange characteristics for the MS profiling of serum samples was published by Callesen et al.52.

2.8 Analysis of cell culture supernatants using magnetic particles

Because of the complex nature of biological fluids such as plasma or serum, biomarker discovery efforts have not as yet delivered any novel single tumor marker 53. Therefore, in the last years the MS or gel electrophoretic analysis of the secretome from specific cancer cell lines, such as pancreatic cancer cells 54, breast cancer cells 55, 56 and also other cell types, e. g. myeloid cells 57 or astrocytes 58, is gaining more and more influence. Due to the low protein concentration and difficulties during the protein isolation from cell culture supernatants, very high numbers of cells (108–109 cells) are normally needed for a successful analysis by MS or gel electrophoresis 54–58. In the last years, novel magnetic particles with hydrophobic characteristics and improved protein binding capacities were developed by Peter et al.59. Using these magnetic RP particles combined with a serum surrogate for an unaltered cell growth of different breast cancer cell lines, peptides could be visualized as low as 10 pM by LC-MALDI-TOF MS. With this strategy (Fig. 1), several differences in the peptide secretion pattern of different progressed breast cancer cell lines were detected 59.

Figure 1.

Flow chart for the isolation, purification and MS detection of substances secreted by carcinoma cells.

3 Concluding remarks

This review is focused on magnetic particles for the enrichment and purification of peptides and proteins prior to their MS or gel electrophoretic analysis. Due to the elegant handling using only magnetic devices and the high binding capacities of magnetic particles with small diameters, the magnetic purification has proven to be a powerful tool for the highly sensitive and rapid detection of peptides and proteins from different human fluids especially by MS. Due to the magnetic core, the sample preparation can be also automated using robot systems, resulting in minimized handling errors during the protein purification process as compared with other purification procedures, such as protein precipitation. With the increasing implementation of magnetic particles with novel surface textures and enhanced protein binding capacities, the investigation of serum or plasma by proteomic methods can be also extended to cell culture supernatants of single cell types. The investigation of supernatants originating from defined cell types avoids the complexity of serum or plasma and high abundant proteins, such as albumin. This opens the possibility for the identification of low concentrated and hitherto unknown tumor markers.

Acknowledgements

This review is dedicated to my beloved brother, Stefan Peter, who died at the age of 49, on September 20, 2009.

The authors have declared no conflict of interest.

Biographical Information

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Jochen F. Peter graduated from the chemistry department, University of Constance in 1994. From 1995-1998 he performed his Ph.D. thesis in the field of biomarker development at the Technical University of Munich working together with Boehringer Mannheim GmbH at Penzberg, Germany. From 1999 to 2000 he held a NIH scholarship at the National Institute of Environmental Health Sciences at Raleigh, USA. From 2004 to mid 2009, he was project leader in the field of cell secretion analysis and protein purification between the Technical University of Munich and Roche Diagnostics GmbH at Penzberg, Germany. In September 2009 he founded the company SensScreen Technologies GmbH at Esslingen, Germany. In April 2007 he won the ABRF Travel Award for Outstanding Scientists and Technologists and in October 2007 the Young Investigator Award of the Human Proteome Organization (HUPO).

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