AnchorChip

A Novel Approach Using MALDI-TOF/TOF Mass Spectrometry and Prestructured Sample Supports (AnchorChip Technology) for Proteomic Profiling and Protein Identification... 

Key Words MALDI TOF/TOF profiling biomarkers AnchorChip tandem MS magnetic beads clinical proteomics serum. 

Matrix/Analyte Dilution and Spotting Onto AnchorChip Target... 

AnchorChip target, 384 spots with 600 pm anchors (Bruker Daltonics, see Note 4)... 

Mix 1 pL of eluted sample and 9 pL matrix solution (from Subheading 3.1.) and spot 1 pL onto AnchorChip target. Continue spotting the rest of the mixture onto the target ( 8 spots). Let the samples dry at room temperature (see Note 6). 

Dissolve the dried peptide calibration standards by adding 125 pL 0.1% TFA to the tube (final concentration will be 4 pmol/pL for each peptide). Mix 1 pL of peptide standard and 9 pL matrix solution and spot 1 pL onto the calibration spots of the AnchorChip target. The final concentration of peptide standard on the target is 400 fmol of each peptide. 

The AnchorChip target is reusable. We clean the target using the following... 

In the case of MALDl-MS, the nanoHPLC has to be coupled to the mass spectrometer offline. After separation, the sample is transferred to a sample plate (target), e.g. AnchorChip target (Broker Daltonics, Bremen, Germany), equipped with a special coating that provides improved crystallization of M ALDI samples and a 10- to 100-fold sensitivity increase. Small hydrophilic positions/anchors (600 pm diameter) are applied equidistantly on the hydrophobic surface of the AnchorChip target thereby reducing sample spreading to only a small area. 

This results in co-crystaUization of matrix and sample and therefore higher sample concentration focused at the distinct hydrophilic anchor points [16]. One of the most commonly used matrices is a saturated solution of a-cyano-4-hydroxy-cinnamic acid in 97% acetone/3% water containing a small amoimt of TFA to support the ionization of the sample molecules in the MALDI source. The matrix is directed to the AnchorChip target and this is then dried. The sample fractionation after HPLC can be performed automatically using special sample fractionation systems (e.g., Probot, LC Packings Dionex). The target is introduced to a special desk and can be adjusted in the three XYZ planes so that the small nano-LC volume is placed directly onto the matrix. Using this set-up, fractions of volume less than 40 nL can be collected automatically. After collection, the individual fractions are recrystallized from 0.01% TFA, 30% acetone, and 60% ethanol in water and analyzed. 

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