poster number mp 565

3 Dimensional MALDI Plates employing collimated-hole structures used to coupling high capacity, high flow separations to MALDI-TOF analysis for top down proteomics Poster Number MP 565

Stephen Hattan, Marvin Vestal Virgin Instruments, Sudbury,

MAINTRODUCTION

-Collimated-hole structures are used to construct 3 dimensional MALDI plates1

- Individual holes filled with monolithic chromatography media -Styrene/divinylbenzene and Butyl and Stearyl methacrylate for reversedphase (RP) capture -Glycidyl methacrylate2 and vinyl azalactone3 co-polymers for immobilized enzyme plates

-3D plates are envisioned to enable high capacity (1mg) loading and high flow rate chromatography (200μL/min - 1mL/min) directly to MALDI-MS and MS-MS analysis-Top down proteomic workflows employing serial and parallel digestion of sample presented

MATERIALS & METHODS

3D MALDI PLATES-Current plates are constructed by machining holes into large format (4.875 x 5.000 x 0.125in) MALDI plates designed for Virgin Instruments mass spectrometer

--although plates may be formatted to any dimensions-Conical holes designed to maximize capacity for sample capture and minimize non-conductive polymer surface on analytical plate surface -variety of polymers have been constructed for RP peptide captureusing hardware designed for either UV and thermal initiation-sample is loaded separately and sequentially into the individual holes on CHS plate but sample elution and washing (if necessary)takes place simultaneously-sample are loaded through the analyticalplate surface (small hole) and eluted inthe opposite direction with matrix

-

TOP DOWN PROTEOMICS WITH SERIAL DIGESTION

-2D LC used to separate protein sample1) RP capture media in plate allows for high-resolution RP separation in 1st dimension

2) Anion exchange 2nd dimensioncompatible in-line trypsin digestion column

3) Digested peptides captured directly onto MALDI plate

4)Sample washed and eluted to surface forMALDI analysis

5)Plate is dried and analyzed By MALDI MS

6) Resulting peptide used to IDprotein by peptide mass fingerprinting or MS-MS analysis used for sequencing and database search

7) Trial experimentation has collected data from 500μg of collected in 30 1st dimension RP frxsSeparated and digested in series and spotted on asingle MALDI plate

C4 separation of Muscle extract

RP fractions further separated by Anion Ex.

Immobilized Enzyme ColumnO-ring seal to plate

Excess solvent passes through while peptides are

captured

10mm trypsin plate

Proteins loadedDigestedPeptides Captured

Excess solvent

1.5mm RP capture plate

0.3mm teflon gasket

wash

Sample eluted to surface

with matrix

Analysis

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1439.77311478.7500

1479.7950

1519.7996

1537.8140

1567.7682

1627.8699

1639.9668 1724.9076

1881.0930

1882.4622

1500 1600 1700 1800 1900

0.000

0.005

0.010

0.015

Daltons

Volts

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1479.7950

1519.8542

1567.7743

1640.00051724.9205

1881.13582274.7887

1500 2000

0.000

0.005

0.010

Daltons

Volts

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1479.7950

1520.8232

1567.7411

1881.1775

1500 2000

0.000

0.001

0.002

0.003

Daltons

Volts

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1439.7731

1479.7950

1519.7996

1537.8140

1567.7682

1627.8699

1639.9668 1724.9076

1881.0930

1921.1660 1995.23752274.8059

1500 2000

0.000

0.005

0.010

0.015

Daltons

Volts

0

20000

40000

60000

80000

100000

120000

1 11 21 31 41

well number

TIC

13

00

-25

00

Da

TOP DOWN PROTEOMICS WITH PARALLEL DIGESTION

Individual samples or a single sample separatedby 1 or more dimensionsof chromatography may be digested in parallel using a CHS immobilized enzyme (IE) plate coupled to a RP capture plate

1)IE and RP plates with Teflon gasket are bolted together2) Protein sample in passed through holes for digest and peptide capture 3) Plates are separated and peptides are eluted to RP plate surface for MS4) Results show TIC (1200-2500) and low, median and high spectra from the parallel digestion of 50 BSA samples

Parallel Digestion of 50 BSA SAMPLES

Funding:This work was supported by National Institute of Health SBIR Grant GM079833

References:1) Hattan SJ, Vestal ML Anal. Chem., 2008, 80 (23), pp 9115–91232) D. S. Peterson, T. Rohr, F. Svec, J. M. J. Fréchet, Anal. Chem., 2002, 74, pp 4081-4088 3) G. T. Hermanson, A. K. Mallia, P. K. Smith, Immobilized Affinity Ligand Techniques Academic Press Inc.

MS Analysis off of polymersurface is detrimental to signal resolution

TIC

12

00

-25

00

Well