Magnetic Sample Preparation For Mass SpectrometryStine Bergholtz, Elisabeth Breivold, David Gillooly, Kirsten Lycke, Pål Songe, Geir Fonnum, Johanna Steen* & Marie Bosnes, Dynal Biotech ASA, Molecular Systems R&D, P.O.Box 114 Smestad, N-0309 Oslo, Norway. *Royal Institute of Technology, Albanova University Center (KTH) Department of Biotechnology, Stocholm, Sweden.

Desalting before MS analysisBefore MS analysis it is important to remove contaminants like surfactants, polymers and salts. Dynabeads® TALON™ were used followed by reversed phase separation with Dynabeads® RPS-PRO to purify and desalt histidine tagged proteins for MS analysis. This protocol has been fully automated on the Magnetic Biosolutions Magnatrix™ 1200 Workstation.

Mass spectrometry spectra and reconstructed (deconvoluted) mass diagram on purifi ed and desalted Mass spectrometry spectra and reconstructed (deconvoluted) mass diagram on purifi ed and desalted Mass spectrometry spectra and reconstructed (deconvoluted) mass diagram on purifi ed and desalted Mass spectrometry spectra and reconstructed (deconvoluted) mass diagram on purifi ed and desalted polyhistidine tagged protein. A: Raw TOF MS spectrum (single scan) of the polyhistidine tagged protein after bead cleanup. B: Reconstructed spectrum obtained from A by MaxEnt1 deconvolution. MS conditions: Micromass Q-TOF 2, positive ion mode, nanofl ow ESI source, direct infusion via syringe pump at 200 µl/min, 2.5 s/scan, 0,1 s interscan delay. 10 µl approx. 17 pM protein solution was injected.

Sample complexity can be reduced by fractionation using Dynabeads Weak Cation Exchange (WCX) or Dynabeads® or Dynabeads® or Dynabeads Reversed Phase Separation (RPS-PRO). Complex samples can be fractionated by either altering the binding conditions in separate reactions or by manipulating elution conditions. This can be achieved by eluting in separate reactions or by eluting step-wise from the same beads.

An outline of the use of Dynabeads TALON™ and Dynabeads RPS-PRO for purifi cation and desalting of polyhistidine tagged recombinant proteins for MS analysis. The polyhistidine tagged recombinant protein immobilised on the beads can be used for protein complex capture. The TALONTMprotein immobilised on the beads can be used for protein complex capture. The TALONTMprotein immobilised on the beads can be used for protein complex capture. The TALON chemistry is licensed from BD Biosciences Clontech.

IntroductionThe complex nature of biological samples and large variations in protein abundance, coupled with dynamic range constraints of downstream analytical techniques (e.g. Mass Spectrometry and 2D gels), make it often necessary to reduce sample complexity for many proteomics strategies, notably biomarker discovery and clinical proteomics. However, sample preparation can become a bottleneck. The use of magnetic beads for sample preparation enables protocols to be automated and throughput to be increased.

We have developed 1 µm Dynabeads® TALONTM for the isolation of recombinant polyhistidine tagged proteins and Reversed Phase Separation (RPS-PRO) and Weak Cation Exchange (WCX) magnetic beads for the fractionation of complex protein samples. We show here that protocols implementing the use of these beads can address some of the most pressing sample preparation problems associated with proteomics.

ConclusionDynabeads® are uniform, superparamagnetic, monodisperse beads with a specifi c and defi ned surface for the adsorption or coupling of bioreactive molecules. The uniform monodisperse nature of Dynabeads® facilitates reproducible sample preparation procedures. High quality MS spectra can be obtained when isolating recombinant polyhistidine tagged proteins using Dynabeads® TALONTM followed by a desalting step using Dynabeads® RPS-PRO. This provides a rapid and reliable procedure for QC testing of recombinant proteins or for the isolation of protein complexes. Sample complexity can be reduced by fractionating complex mixtures using Dynabeads® RPS-PRO and Dynabeads® WCX. Rapid kinetics and ease of automation of procedures using magnetic beads enables highly increased throughput of analyses. The Dynabead® technology offers a fl exible, scalable and gentle tool for sample handling and complexity reduction for proteomic research, systems biology and drug discovery.

Protein mixtures can be fractionated using Dynabeads® Reverse Phase Separation (RPS-PRO)

Results: Which proteins are eluted depends upon the concentration of acetonitrile used for elution. This means that protein mixtures can be fractionated using Dynabeads® RPS-PRO.

Dynabeads®Dynabeads®Dynabeads RPS-PRO produce concentrated desalted protein samples/fractions that can subsequently be applied to an MS instrument. A mixture of fi ve proteins with differing characteristics was adsorbed to Dynabeads®Dynabeads®Dynabeads RPS-PRO in a low salt buffer containing trifl uoroacetic acid (TFA) as an ion-pairing agent. Following a washing step these proteins were desorbed in 0, 15, 30, 40, or 50% acetonitrile and the resulting eluates examined by SDS-PAGE gel electrophoresis (Bio-Rad Criterion 10 - 20 % Tris HCl gels). Gels were silver stained.

Marker Elution in ACN (%)

0 15 30 40 50

Protein Mix

Conalbumin

Diamine Oxidase

Alcohol DH

Cyt C

Trp Inhibitor

SEM picture of Dynabeads® MyOneTM

An outline of the use of Dynabeads®An outline of the use of Dynabeads®An outline of the use of Dynabeads TALON™ and Dynabeads® TALON™ and Dynabeads® TALON™ and Dynabeads RPS-PRO for purifi cation and desalting

96-well culture plate with tagged protein

Dynabeads® TALON™

Cell lysis Washing

Elution+

Tagged proteinimmobilised on bead

Elution

Purified protein complex

Dynabeads® RPS-PRO

Purified proteinfor MS analysis

ElutionDesalting

Capture of recombinant proteins for MS analysis

DeconvolutionDeconvolutionDeconvolutionDeconvolutionDeconvolutionDeconvolutionDeconvolutionDeconvolutionDeconvolutionDeconvolutionDeconvolution

Fractionation using Dynabeads® Weak Cation Exchange (WCX) reduces sample complexity

Results: The three fractions analysed enabled the detection of 2393 unique resolved spots. Of these only 121 spots were found to be present in all three fractions (5%).

A lysate from a Human cancer cell line (SW480) was fractionated using Dynabeads®was fractionated using Dynabeads®was fractionated using Dynabeads WCX. Proteins were adsorbed to Dynabeads®were adsorbed to Dynabeads®were adsorbed to Dynabeads WCX using low salt conditions (50 mM sodium phosphate buffer pH 7.0, 50 mM NaCl, 0.01 % Tween 20), the beads washed, and proteins then eluted step-wise in buffers containing increasing salt concentrations (50 mM sodium phosphate buffer pH 7.0, 200 mM – 1 M NaCl, 0.01 % Tween 20).

Three of the resulting fractions (200 mM, 400 mM & 600 mM NaCl) were analysed by 2D gel electrophoresis. Fractions were prepared for gel analysis by desalting using a Bio-Rad Ready Prep 2D clean up kit followed by rehydration in Amersham DeStreak Rehydration Solution containing Amersham DeStreak Reagent. Samples were then focused in Bio-Rad ReadyStrip pH 3 – 10 (11 cm) IPG strips and resolved in Bio-Rad Criterion 8 – 16 % SDS-PAGE gels. Approximately 50 µg protein was applied to each strip. Gels were silver stained and analysed using PDQuest 2D gel analysis software from Bio-Rad.

pH 3pH 3 pH 10pH 10

200 mM fraction

910 spots, 78% unique to this fraction

400 mM fraction

1254 spots, 66% unique to this fraction

600 mM fraction

652 spots, 67% unique to this fraction

Sample complexity can be reduced by fractionation using Dynabeads®Sample complexity can be reduced by fractionation using Dynabeads®Sample complexity can be reduced by fractionation using Dynabeads Weak Cation Exchange (WCX)

WCXIncreasingSalt/pH

Cell lysate

Protein/ peptide mix

WCX /RPS-PRObeads (1�m)

RPS-PRO

IncreasingAcetonitrile

Step-Eluted proteins/peptides

Reduction of sample complexity by protein fractionation

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Summary

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