Poster Number MP 565
1
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, MA INTRODUCTION -Collimated-hole structures are used to construct 3 dimensional MALDI plates 1 - Individual holes filled with monolithic chromatography media -Styrene/divinylbenzene and Butyl and Stearyl methacrylate for reversed phase (RP) capture -Glycidyl methacrylate 2 and vinyl azalactone 3 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 capture using 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 analytical plate surface (small hole) and eluted in the opposite direction with matrix TOP DOWN PROTEOMICS WITH SERIAL DIGESTION -2D LC used to separate protein sample 1) RP capture media in plate allows for high-resolution RP separation in 1 st dimension 2) Anion exchange 2 nd dimension compatible in-line trypsin digestion column 3) Digested peptides captured directly onto MALDI plate 4)Sample washed and eluted to surface for MALDI analysis 5)Plate is dried and analyzed By MALDI MS 6) Resulting peptide used to ID protein 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 1 st dimension RP frxs Separated and digested in series and spotted on a single MALDI plate C4 separation of Muscle extract RP fractions further separated by Anion Ex. Immobilized Enzyme Column O-ring seal to plate Excess solvent passes through while peptides are captured 10mm trypsin plate Proteins loaded Digested Peptides Captured Excess solvent 1.5mm RP capture plate 0.3mm teflon gasket wash Sample eluted to surface with matrix Analysi s \\domainserver\public\Shared Spectra\Steve Sept2008\steve_9_18\MiniTof-2008_09_18_09_03\M iniTof-0017-2008_09_18_10_03_50.578-R un1.m zm l#14 1439.7731 1478.7500 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 D altons V olts \\dom ainserver\public\Shared Spectra\Steve Sept2008\steve_9_18\M iniTof-2008_09_18_09_03\M iniTof-0001-2008_09_18_09_08_12.858-R un1.m zm l#18 1479.7950 1519.8542 1567.7743 1640.0005 1724.9205 1881.1358 2274.7887 1500 2000 0.000 0.005 0.010 D altons Volts \\dom ainserver\public\Shared Spectra\Steve Sept2008\steve_9_18\M iniTof-2008_09_18_09_03\M iniTof-0003-2008_09_18_09_10_06.901-R un1.m zm l#5 1479.7950 1520.8232 1567.7411 1881.1775 1500 2000 0.000 0.001 0.002 0.003 D altons Volts \\dom ainserver\public\Shared Spectra\Steve Sept2008\steve_9_18\M iniTof-2008_09_18_09_03\M iniTof-0017-2008_09_18_10_03_50.578-R un1.m zm l#14 1439.7731 1479.7950 1519.7996 1537.8140 1567.7682 1627.8699 1639.9668 1724.9076 1881.0930 1921.1660 1995.2375 2274.8059 1500 2000 0.000 0.005 0.010 0.015 D altons Volts 0 20000 40000 60000 80000 100000 120000 1 11 21 31 41 w ellnum ber TIC 1300-2500 TOP DOWN PROTEOMICS WITH PARALLEL DIGESTION Individual samples or a single sample separated by 1 or more dimensions of 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 together 2) Protein sample in passed through holes for digest and peptide capture 3) Plates are separated and peptides are eluted to RP plate surface for MS 4) Results show TIC (1200-2500) and low, median and high spectra from the parall 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 9115–9123 2) 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 polymer surface is detrimental to signal resolution TIC 1200- 2500 Well
description
3 Dimensional MALDI Plates employing collimated-hole structures used to coupling high capacity, high flow separations to MALDI-TOF analysis for top down proteomics. Proteins loaded Digested Peptides Captured. 10mm trypsin plate. 0.3mm teflon gasket. Excess solvent. 1.5mm RP capture plate. - PowerPoint PPT Presentation
Transcript of 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
\\domainserver\public\Shared Spectra\Steve Sept 2008\steve_9_18\MiniTof-2008_09_18_09_03\MiniTof-0017-2008_09_18_10_03_50.578-Run1.mzml#14
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
\\domainserver\public\Shared Spectra\Steve Sept 2008\steve_9_18\MiniTof-2008_09_18_09_03\MiniTof-0001-2008_09_18_09_08_12.858-Run1.mzml#18
1479.7950
1519.8542
1567.7743
1640.00051724.9205
1881.13582274.7887
1500 2000
0.000
0.005
0.010
Daltons
Volts
\\domainserver\public\Shared Spectra\Steve Sept 2008\steve_9_18\MiniTof-2008_09_18_09_03\MiniTof-0003-2008_09_18_09_10_06.901-Run1.mzml#5
1479.7950
1520.8232
1567.7411
1881.1775
1500 2000
0.000
0.001
0.002
0.003
Daltons
Volts
\\domainserver\public\Shared Spectra\Steve Sept 2008\steve_9_18\MiniTof-2008_09_18_09_03\MiniTof-0017-2008_09_18_10_03_50.578-Run1.mzml#14
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
