MALDI MS peptide mapping performance by in-gel digestion on a probe with prestructured sample supports

Tina Guldberg Klenø, Christian Maaløv Andreasen, Helle Ørsted Kjeldal, Lise Rønnedal Leonardsen, Thomas Nylandsted Krogh, Per Franklin Nielsen, Marianne Vind Sørensen, Ole Nørregaard Jensen

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Matrix-assisted laser desorption/ionization (tandem) mass spectrometry (MALDI MS) is widely used in protein chemistry and proteomics research for the identification and characterization of proteins isolated by polyacrylamide gel electrophoresis. In an effort to minimize sample handling and increase sample throughput, we have developed a novel in-gel digestion protocol where sample preparation is performed directly on a MALDI probe with prestructured sample support. The protocol consists of few sample-handling steps and has minimal consumption of reagents, making the protocol sensitive, timesaving, and cost-efficient. Performance of the on-probe sample preparation protocol was demonstrated by analysis of a set of rat liver proteins obtained from a fluorescently stained (Cy3 and SyproRuby) two-dimensional polyacrylamide gel. The success rate of protein identification by on-probe tryptic digestion and MALDI peptide mass mapping was 89%. The on-probe in-gel digestion procedure provided superior sensitivity and peptide mass mapping performance as compared to our standard in-gel digestion protocol. The on-probe digestion technique resulted in significantly improved amino acid sequence coverage of proteins, mainly due to efficient recovery and detection of large (>1.5 kDa) hydrophobic peptides. These observations indicate that numerous tryptic peptides are lost when using the standard in-gel digestion methods and sample preparation techniques for MALDI MS. This study also demonstrates that the on-probe digestion protocol combined with MALDI tandem mass spectrometry provides a robust platform for proteomics research, including protein identification and determination of posttranslational modifications.
Original languageEnglish
JournalAnalytical Chemistry
Volume76
Issue number13
Pages (from-to)3576-83
Number of pages7
ISSN0003-2700
DOIs
Publication statusPublished - 1. Jul 2004

Fingerprint

Ionization
Mass spectrometry
Desorption
Gels
Peptides
Lasers
Proteins
Electrophoresis
Liver
Rats
Throughput
Amino Acids
Recovery
Costs
Proteomics
polyacrylamide gels

Keywords

  • Animals
  • Cattle
  • Electrophoresis, Gel, Two-Dimensional
  • Gels
  • Liver
  • Molecular Probes
  • Peptide Mapping
  • Proteins
  • Rats
  • Sensitivity and Specificity
  • Serum Albumin, Bovine
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Cite this

Klenø, T. G., Andreasen, C. M., Kjeldal, H. Ø., Leonardsen, L. R., Krogh, T. N., Nielsen, P. F., ... Jensen, O. N. (2004). MALDI MS peptide mapping performance by in-gel digestion on a probe with prestructured sample supports. Analytical Chemistry, 76(13), 3576-83. https://doi.org/10.1021/ac0499120
Klenø, Tina Guldberg ; Andreasen, Christian Maaløv ; Kjeldal, Helle Ørsted ; Leonardsen, Lise Rønnedal ; Krogh, Thomas Nylandsted ; Nielsen, Per Franklin ; Sørensen, Marianne Vind ; Jensen, Ole Nørregaard. / MALDI MS peptide mapping performance by in-gel digestion on a probe with prestructured sample supports. In: Analytical Chemistry. 2004 ; Vol. 76, No. 13. pp. 3576-83.
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abstract = "Matrix-assisted laser desorption/ionization (tandem) mass spectrometry (MALDI MS) is widely used in protein chemistry and proteomics research for the identification and characterization of proteins isolated by polyacrylamide gel electrophoresis. In an effort to minimize sample handling and increase sample throughput, we have developed a novel in-gel digestion protocol where sample preparation is performed directly on a MALDI probe with prestructured sample support. The protocol consists of few sample-handling steps and has minimal consumption of reagents, making the protocol sensitive, timesaving, and cost-efficient. Performance of the on-probe sample preparation protocol was demonstrated by analysis of a set of rat liver proteins obtained from a fluorescently stained (Cy3 and SyproRuby) two-dimensional polyacrylamide gel. The success rate of protein identification by on-probe tryptic digestion and MALDI peptide mass mapping was 89{\%}. The on-probe in-gel digestion procedure provided superior sensitivity and peptide mass mapping performance as compared to our standard in-gel digestion protocol. The on-probe digestion technique resulted in significantly improved amino acid sequence coverage of proteins, mainly due to efficient recovery and detection of large (>1.5 kDa) hydrophobic peptides. These observations indicate that numerous tryptic peptides are lost when using the standard in-gel digestion methods and sample preparation techniques for MALDI MS. This study also demonstrates that the on-probe digestion protocol combined with MALDI tandem mass spectrometry provides a robust platform for proteomics research, including protein identification and determination of posttranslational modifications.",
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author = "Klen{\o}, {Tina Guldberg} and Andreasen, {Christian Maal{\o}v} and Kjeldal, {Helle {\O}rsted} and Leonardsen, {Lise R{\o}nnedal} and Krogh, {Thomas Nylandsted} and Nielsen, {Per Franklin} and S{\o}rensen, {Marianne Vind} and Jensen, {Ole N{\o}rregaard}",
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Klenø, TG, Andreasen, CM, Kjeldal, HØ, Leonardsen, LR, Krogh, TN, Nielsen, PF, Sørensen, MV & Jensen, ON 2004, 'MALDI MS peptide mapping performance by in-gel digestion on a probe with prestructured sample supports', Analytical Chemistry, vol. 76, no. 13, pp. 3576-83. https://doi.org/10.1021/ac0499120

MALDI MS peptide mapping performance by in-gel digestion on a probe with prestructured sample supports. / Klenø, Tina Guldberg; Andreasen, Christian Maaløv; Kjeldal, Helle Ørsted; Leonardsen, Lise Rønnedal; Krogh, Thomas Nylandsted; Nielsen, Per Franklin; Sørensen, Marianne Vind; Jensen, Ole Nørregaard.

In: Analytical Chemistry, Vol. 76, No. 13, 01.07.2004, p. 3576-83.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - MALDI MS peptide mapping performance by in-gel digestion on a probe with prestructured sample supports

AU - Klenø, Tina Guldberg

AU - Andreasen, Christian Maaløv

AU - Kjeldal, Helle Ørsted

AU - Leonardsen, Lise Rønnedal

AU - Krogh, Thomas Nylandsted

AU - Nielsen, Per Franklin

AU - Sørensen, Marianne Vind

AU - Jensen, Ole Nørregaard

PY - 2004/7/1

Y1 - 2004/7/1

N2 - Matrix-assisted laser desorption/ionization (tandem) mass spectrometry (MALDI MS) is widely used in protein chemistry and proteomics research for the identification and characterization of proteins isolated by polyacrylamide gel electrophoresis. In an effort to minimize sample handling and increase sample throughput, we have developed a novel in-gel digestion protocol where sample preparation is performed directly on a MALDI probe with prestructured sample support. The protocol consists of few sample-handling steps and has minimal consumption of reagents, making the protocol sensitive, timesaving, and cost-efficient. Performance of the on-probe sample preparation protocol was demonstrated by analysis of a set of rat liver proteins obtained from a fluorescently stained (Cy3 and SyproRuby) two-dimensional polyacrylamide gel. The success rate of protein identification by on-probe tryptic digestion and MALDI peptide mass mapping was 89%. The on-probe in-gel digestion procedure provided superior sensitivity and peptide mass mapping performance as compared to our standard in-gel digestion protocol. The on-probe digestion technique resulted in significantly improved amino acid sequence coverage of proteins, mainly due to efficient recovery and detection of large (>1.5 kDa) hydrophobic peptides. These observations indicate that numerous tryptic peptides are lost when using the standard in-gel digestion methods and sample preparation techniques for MALDI MS. This study also demonstrates that the on-probe digestion protocol combined with MALDI tandem mass spectrometry provides a robust platform for proteomics research, including protein identification and determination of posttranslational modifications.

AB - Matrix-assisted laser desorption/ionization (tandem) mass spectrometry (MALDI MS) is widely used in protein chemistry and proteomics research for the identification and characterization of proteins isolated by polyacrylamide gel electrophoresis. In an effort to minimize sample handling and increase sample throughput, we have developed a novel in-gel digestion protocol where sample preparation is performed directly on a MALDI probe with prestructured sample support. The protocol consists of few sample-handling steps and has minimal consumption of reagents, making the protocol sensitive, timesaving, and cost-efficient. Performance of the on-probe sample preparation protocol was demonstrated by analysis of a set of rat liver proteins obtained from a fluorescently stained (Cy3 and SyproRuby) two-dimensional polyacrylamide gel. The success rate of protein identification by on-probe tryptic digestion and MALDI peptide mass mapping was 89%. The on-probe in-gel digestion procedure provided superior sensitivity and peptide mass mapping performance as compared to our standard in-gel digestion protocol. The on-probe digestion technique resulted in significantly improved amino acid sequence coverage of proteins, mainly due to efficient recovery and detection of large (>1.5 kDa) hydrophobic peptides. These observations indicate that numerous tryptic peptides are lost when using the standard in-gel digestion methods and sample preparation techniques for MALDI MS. This study also demonstrates that the on-probe digestion protocol combined with MALDI tandem mass spectrometry provides a robust platform for proteomics research, including protein identification and determination of posttranslational modifications.

KW - Animals

KW - Cattle

KW - Electrophoresis, Gel, Two-Dimensional

KW - Gels

KW - Liver

KW - Molecular Probes

KW - Peptide Mapping

KW - Proteins

KW - Rats

KW - Sensitivity and Specificity

KW - Serum Albumin, Bovine

KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

U2 - 10.1021/ac0499120

DO - 10.1021/ac0499120

M3 - Journal article

VL - 76

SP - 3576

EP - 3583

JO - Analytical Chemistry

JF - Analytical Chemistry

SN - 0003-2700

IS - 13

ER -

Klenø TG, Andreasen CM, Kjeldal HØ, Leonardsen LR, Krogh TN, Nielsen PF et al. MALDI MS peptide mapping performance by in-gel digestion on a probe with prestructured sample supports. Analytical Chemistry. 2004 Jul 1;76(13):3576-83. https://doi.org/10.1021/ac0499120