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-Aluminum housing with o-ring seal allows for washing
1
6 106 20 40 60 80 Y P osition -Aluminum housing with o-ring seal allows for washing (salt removal) , plate cleaning and regeneration enabling for multiple use -Sample elution for MS analysis accomplished by forcing matrix in organic solution through one side of the plate and then drying eluent on the opposite side High resolution MALDI plates for the direct coupling of PAGE separations and tissue analysis with MALDI mass spectrometry WP27 599 Stephen J. Hattan ; Kenneth C. Parker ; Marvin L. Vestal VIC Instruments Corporation, Sudbury , MA Methods Plate Construction -Micro channel plates piranha solution cleaned and silanized (2% methylacryloxypropyl-trimethoxysilane, 95% ethanol) -Two phase polymer plate construction done in teflon mold with UV polymer initiation. Solid outer methacrylate border, Porous inner butylmethacrylate monolith 2 active surface (40% polymer, 60 % porogen, 0.4% initiator) - u channel plate teflon mold Solid methacrylate border Porous polymer monolith nless steel plates block UV light during solid der construction lates removed for interior monolith construction ss polymer is removed with a razor blade Protein / peptide migration + - PAGE-Gel Interface -CHS plate takes place of peptide capture membrane in “Molecular Scanner” 3,4 -PAGE separated protein is blotted through trypsin membrane and captured on CHS plate PAGE Trypsin CHS Capture Gel membrane plate Collimated-Hole Structure (CHS) Capture Plate Polymer monolith Solid methacrylate border 35um 25m ~10,000,000 holes Peptide elution and plate washing / cleaning Introduction High-resolution, 3-dimensional MALDI-TOF plates 1 developed as a direct interface between PAGE separations and tissue samples with MALDI mass spectrometry. Construction uses µ-channel plates composed of 25 µm ID collimated-holes structures (CHS) filled with monolithic chromatography media. Plates operate by capturing and concentrating sample (protein / peptides) in the porous-structured, hydrophobic plate interior. After capture, material is eluted back to the surface using organic solvents containing MALDI matrix. Upon drying, analytes are incorporated into matrix crystals on the plate surface. Finally, the plate is loaded directly into the mass spectrometer for analysis. 1475.7 1600.87 1592.9 1271.6 84 rows, 3 columns PMF Search Conditions Mass Spectrometry • Ion Current plotted below Vs. Spot Position. • 18195 spectra of 200 laser shots. • kHz laser, scanning 1.2 mm / sec. • m/z range 150 – 3000. • 0.2 mm per spectrum in y (direction of electrophoresis). • 1 mm per spectrum in x. • All Spectra mapped to a spot, 1 mm x 35 mm. • -> 252 averaged spectra ( 84 rows, 3 columns). Distribution of total ion current Colored heat map: Distribution of 4 masses across the plate Conclusions •Can couple SDS gels to monolithic capture plates. •Can elute peptides to surface using matrix solution. •Can identify multiple proteins per SDS gel band. Spectrum A data Spectrum B data Symbol key Results References 1) Hattan SJ, Vestal ML (2008) Anal Chem.; 80 : 9115-9123. 2) D. S. Peterson, T. Rohr, F. Svec, J. M. J. Fréchet (2002) Anal. Chem.; 74 : 4081-4088. 3) Binz PA et al. (1999) Anal. Chem.; 71 : 4981-4988. 4) Nadler TK, et al. (2004) Anal. Biochem.; 332 : 337-348. 5) Parker KC (2002) Scoring Methods in MALDI Peptide Mass Fingerprinting.. JASMS; 13 : 22-39. Funding: SBIR Grant GM079833 Plate with matrix 6 106 20 40 60 80 Y P osition 1300 -2600 sym b IR ank Sequence mz Int Ch ppm MB 1 VEADVAGHGQEVLLR 1592.8 636 20.0 0.2 MB 2 LFTG HPETLEK 1271.7 215 1.8 -1.5 MB 2a HLAESHANKHK 1271.7 215 0.4 0.7 MB 4 GHHEAEVK 906.4 151 2.0 4.6 HBB 5 LLGNVLVVVLAR 1265.8 132 20.0 -3.2 HBB 6 VVAGVANALAHR 1177.7 114 20.0 1.1 HBB 11 VKVDEVGGEALGR 1328.7 46 7.0 0.7 HBB 70 VVAGVANALAHRYH 1477.8 2 2.0 12.6 MB 74 HG NTVLTALG G LLK 1393.8 1 2.0 -7.4 peptide data sym b IR ank Sequence mz Int Ch ppm TPM 2 2.0 KLVLLEGELER 1298.8 376 6.0 5.8 ALDOA 3.0 ADDGRPFPQVLK 1342.7 251 20.0 -0.1 ALDOA 6.0 YSHEELAm ATVTALR 1707.8 178 20.0 -19.7 ALDOA 8.0 RLQSLGTENTEENR 1646.8 111 6.7-16.2 ALDOA 8a LQSLGTENTEENRR 1646.8 111 3.9-16.2 TPM 2 12.0 KYEEVAR 894.5 34 10.5 -14.5 TPM 2 12a YEEVARK 894.5 34 2.0-14.5 ALDOA 13.0 AAQEEYVK 937.5 33 2.0-12.6 TPM 2 14.0 LVLLEG ELER 1170.7 30 18.0 6.4 TPM 2 18.0 ATDAEADVASLNRR 1488.7 25 2.0 4.3 ACTA2 21.0 G YSFVTTAER 1130.5 22 16.2 0.3 TPM 2 37.0 RLQ LVEEELDR 1399.8 6 5.3-10.3 ALDOA 39.0 AAQEEYVKR 1093.6 6 2.0 -0.9 ACTA2 41.0 QEYDEAGPSLVHR 1500.7 5 20.0 3.6 ALDOA 49.0 Q LLLTA D D R 1044.6 3 16.0 -10.6 TPM 2 55.0 TLDDLEDEVYAQKm K 1813.9 3 0.4 13.2 ACTA2 56.0 A G FA G D D A PR 976.4 3 20.0 5.3 TPM 2 62.0 LD K EN A LD R 1073.6 2 6.6 12.2 ALDOA 72.0 zQYVTEKVLAAVYK 1671.9 2 0.2 19.8 TPM 2 72a LVLLEG ELERSEER 1671.9 2 6.8 11.5 ALDOA 86.0 PW ALTFSYG R 1197.6 1 2.0 14.3 ACTA2 111.0 LW HHSFYNELR 1501.7 1 20.0 12.0 peptide data sym b #pep length Sc %ChM % IntM PrR ank ALDOA 9 364 54 33.3 18.9 1 TPM 2 9 284 13 26.5 15.4 2 ACTA2 4 377 7 25.6 1.0 3 protein data sym b #pep length Sc % C hM % IntM PrR ank MB 5 154 490 49.1 44.7 1 HBB 4 145 98 42.4 13.1 2 protein data •MB myoglobin •HBB hemoglobin beta •ALDOA* fructose bisP aldolase •TPM2* tropomyosin •ACTA2* actin • Peptides m /z800-4000 • Proteins 50-3000 aa in length – 16293 cow proteins in all • M atching (C hem Plex,see ref.5) – Tolerance • 4 ppm minimum – (no additional creditppm below this) • <8 ppm (top peak(s)) • <20 ppm (all peaks) – 1 peak in m ostintense 100 w ith C h > 2 – 2 peaks m atched in all – M ore creditw ith intensity – M ore creditform atching expected peptides • % C hS m atched as filter • Arg peptides >> Lys peptides • W eighted scale form issed cleavages • 117 peaks detected IRank Intensity R ank Sc S core Int Intensity % C hM % Chem Score Matched Ch C hem Score % IntM % Intensity M atched # pep # peptides; PrR ank P rotein R ank length #aa in protein A B A B A B in A
description
High resolution MALDI plates for the direct coupling of PAGE separations and tissue analysis with MALDI mass spectrometry. Polymer monolith. -. 35um. +. 25m. Solid methacrylate border. Solid methacrylate border. u channel plate. teflon mold. Porous polymer monolith. WP27 599. - PowerPoint PPT Presentation
Transcript of -Aluminum housing with o-ring seal allows for washing
Area Mean Intensity 26.996 StDev 24.54
6 106 206 306 406
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X Position
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n-Aluminum housing with o-ring seal allows for washing (salt removal) , plate cleaning and regeneration enabling for multiple use
-Sample elution for MS analysis accomplished by forcing matrix in organic solution through one side of the plate and then drying eluent on the opposite side
High resolution MALDI plates for the direct coupling of PAGE separations and tissue analysis with MALDI mass spectrometry WP27 599Stephen J. Hattan ; Kenneth C. Parker; Marvin L. Vestal VIC Instruments Corporation, Sudbury , MA
Methods
Plate Construction-Micro channel plates piranha solution cleaned and silanized (2% methylacryloxypropyl-trimethoxysilane, 95% ethanol)-Two phase polymer plate construction done in teflon mold with UV polymer initiation. Solid outer methacrylate border, Porous inner butylmethacrylate monolith2 active surface (40% polymer, 60 % porogen, 0.4% initiator)
-
u channelplate
teflonmold
Solid methacrylateborder
Porous polymer monolith
-Stainless steel plates block UV light during solid border construction -SS plates removed for interior monolith construction-excess polymer is removed with a razor blade
Protein / peptide migration
+-
PAGE-Gel Interface-CHS plate takes place of peptide capture membrane in “Molecular Scanner”3,4
-PAGE separated protein is blotted through trypsin membrane and captured on CHS plate PAGE Trypsin CHS Capture
Gel membrane plate
Collimated-Hole Structure (CHS) Capture Plate
Polymer monolith
Solid methacrylate border
35um25m
~10,000,000 holes
Peptide elution and plate washing / cleaning
Introduction
High-resolution, 3-dimensional MALDI-TOF plates1 developed as a direct interface between PAGE separations and tissue samples with MALDI mass spectrometry. Construction uses µ-channel plates composed of 25 µm ID collimated-holes structures (CHS) filled with monolithic chromatography media. Plates operate by capturing and concentrating sample (protein / peptides) in the porous-structured, hydrophobic plate interior. After capture, material is eluted back to the surface using organic solvents containing MALDI matrix. Upon drying, analytes are incorporated into matrix crystals on the plate surface. Finally, the plate is loaded directly into the mass spectrometer for analysis.
Area Mean Intensity 15.128 StDev 21.569
6 106 206 306
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X Position
Y P
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1475.71600.871592.91271.6
84 rows, 3 columns
PMF Search ConditionsMass Spectrometry• Ion Current plotted below Vs. Spot Position.• 18195 spectra of 200 laser shots.• kHz laser, scanning 1.2 mm / sec.• m/z range 150 – 3000.• 0.2 mm per spectrum in y (direction of electrophoresis).• 1 mm per spectrum in x.• All Spectra mapped to a spot, 1 mm x 35 mm.• -> 252 averaged spectra ( 84 rows, 3 columns).
Distribution of total ion current
Colored heat map:Distribution of 4 masses across the plate
Conclusions•Can couple SDS gels to monolithic capture plates.•Can elute peptides to surface using matrix solution.•Can identify multiple proteins per SDS gel band.
Spectrum A data
Spectrum B data
Symbol key
Results
References1) Hattan SJ, Vestal ML (2008) Anal Chem.; 80 : 9115-9123.2) D. S. Peterson, T. Rohr, F. Svec, J. M. J. Fréchet (2002) Anal. Chem.; 74 : 4081-4088.3) Binz PA et al. (1999) Anal. Chem.; 71 : 4981-4988.4) Nadler TK, et al. (2004) Anal. Biochem.; 332 : 337-348.5) Parker KC (2002) Scoring Methods in MALDI Peptide Mass Fingerprinting.. JASMS; 13 : 22-39.
Funding: SBIR Grant GM079833Plate with matrix
Area Mean Intensity 284.473 StDev 357.049
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Y P
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n
1300 - 2600
symb IRank Sequence mz Int Ch ppmMB 1 VEADVAGHGQEVLLR 1592.8 636 20.0 0.2MB 2 LFTGHPETLEK 1271.7 215 1.8 -1.5MB 2a HLAESHANKHK 1271.7 215 0.4 0.7MB 4 GHHEAEVK 906.4 151 2.0 4.6HBB 5 LLGNVLVVVLAR 1265.8 132 20.0 -3.2HBB 6 VVAGVANALAHR 1177.7 114 20.0 1.1HBB 11 VKVDEVGGEALGR 1328.7 46 7.0 0.7HBB 70 VVAGVANALAHRYH 1477.8 2 2.0 12.6MB 74 HGNTVLTALGGLLK 1393.8 1 2.0 -7.4
peptide data
symb IRank Sequence mz Int Ch ppmTPM2 2.0 KLVLLEGELER 1298.8 376 6.0 5.8ALDOA 3.0 ADDGRPFPQVLK 1342.7 251 20.0 -0.1ALDOA 6.0 YSHEELAmATVTALR 1707.8 178 20.0 -19.7ALDOA 8.0 RLQSLGTENTEENR 1646.8 111 6.7 -16.2ALDOA 8a LQSLGTENTEENRR 1646.8 111 3.9 -16.2TPM2 12.0 KYEEVAR 894.5 34 10.5 -14.5TPM2 12a YEEVARK 894.5 34 2.0 -14.5ALDOA 13.0 AAQEEYVK 937.5 33 2.0 -12.6TPM2 14.0 LVLLEGELER 1170.7 30 18.0 6.4TPM2 18.0 ATDAEADVASLNRR 1488.7 25 2.0 4.3ACTA2 21.0 GYSFVTTAER 1130.5 22 16.2 0.3TPM2 37.0 RLQLVEEELDR 1399.8 6 5.3 -10.3ALDOA 39.0 AAQEEYVKR 1093.6 6 2.0 -0.9ACTA2 41.0 QEYDEAGPSLVHR 1500.7 5 20.0 3.6ALDOA 49.0 QLLLTADDR 1044.6 3 16.0 -10.6TPM2 55.0 TLDDLEDEVYAQKmK 1813.9 3 0.4 13.2ACTA2 56.0 AGFAGDDAPR 976.4 3 20.0 5.3TPM2 62.0 LDKENALDR 1073.6 2 6.6 12.2ALDOA 72.0 zQYVTEKVLAAVYK 1671.9 2 0.2 19.8TPM2 72a LVLLEGELERSEER 1671.9 2 6.8 11.5ALDOA 86.0 PWALTFSYGR 1197.6 1 2.0 14.3ACTA2 111.0 LWHHSFYNELR 1501.7 1 20.0 12.0
peptide data
symb #pep length Sc %ChM %IntM PrRankALDOA 9 364 54 33.3 18.9 1TPM2 9 284 13 26.5 15.4 2ACTA2 4 377 7 25.6 1.0 3
protein data
symb #pep length Sc %ChM %IntM PrRankMB 5 154 490 49.1 44.7 1HBB 4 145 98 42.4 13.1 2
protein data
•MB myoglobin•HBB hemoglobin beta•ALDOA* fructose bisP aldolase•TPM2* tropomyosin•ACTA2* actin
• Peptides m/z 800-4000• Proteins 50-3000 aa in length
– 16293 cow proteins in all• Matching (ChemPlex, see ref. 5)
– Tolerance • 4 ppm minimum
– (no additional credit ppm below this)• <8 ppm (top peak(s))• <20 ppm (all peaks)
– 1 peak in most intense 100 with Ch > 2 – 2 peaks matched in all– More credit with intensity– More credit for matching expected peptides
• %ChS matched as filter• Arg peptides >> Lys peptides• Weighted scale for missed cleavages
• 117 peaks detected
IRank Intensity Rank Sc ScoreInt Intensity %ChM % ChemScore MatchedCh ChemScore % IntM % Intensity Matched # pep # peptides; PrRank Protein Ranklength #aa in protein
A B
A
B
AB
in A
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