Ion source-dependent performance of 4-vinylpyridine, iodoacetamide, and N-maleoyl derivatives for the detection of cysteine-containing peptides in complex proteomics.
Cysteine is unique among the proteinogenic amino acids due to its ability to form disulfide bonds. While this property is of vital importance for protein structures and biological processes, it causes difficulties for the mass spectrometric identification of cysteine-containing peptides. A common approach to overcome these problems in bottom-up proteomics is the reduction and covalent modification of sulfhydryl groups prior to enzymatic digestion. In this study, established alkylating agents and N-maleoyl amino acids with variable hydrophobicity were characterized with respect to a variety of relevant parameters and subsequently evaluated in a large-scale analysis using different ion sources. Depending on the compound, the ion source had a profound impact on the relative and absolute identification of cysteine-containing peptides. The best results were obtained by derivatization of the cysteine residues with 4-vinylpyridine and subsequent matrix-assisted laser desorption ionization (MALDI). Modification with 4-vinylpyridine increased the number of cysteine-containing peptides identified with any other compound using LC-MALDI/MS at least by a factor of 2. This experimental observation is mirrored by differences in the gas-phase basicities, which were computed for methyl thiolate derivatives of the compounds using density functional theory. With electrospray ionization (ESI), complementary use of reagents from three different compound classes, e.g., iodoacetamide, 4-vinylpyridine, and N-maleoyl beta-alanine, was beneficial compared to the application of a single reagent.
German Cancer Research Center, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.|Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.
Wiebke
W
Nadler
Department of Chemistry, University of Zurich, Winterthurerstr 190, 8057, Zurich, Switzerland.
Regina
R
Berg
German Cancer Research Center, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.|Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.
Philipp
P
Walch
German Cancer Research Center, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.|Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.
Sabrina
S
Hanke
German Cancer Research Center, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.
Mathis
M
Baalmann
German Cancer Research Center, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.|Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.
Alexander
A
Kerner
German Cancer Research Center, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.|Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.
Andreas
A
Trumpp
German Cancer Research Center, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany. c.roesli@dkfz.de.|Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany. c.roesli@dkfz.de.
Christoph
C
Roesli
D000478:Alkylation; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D003545:Cysteine / Q000032:analysis*; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D007460:Iodoacetamide / Q000737:chemistry; D008298:Maleates / Q000737:chemistry; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry*; D040901:Proteomics / Q000379:methods; D011725:Pyridines / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000596:Amino Acids; D029968:Escherichia coli Proteins; D008298:Maleates; D010455:Peptides; D011725:Pyridines; C029351:4-vinylpyridine; D003545:Cysteine; D007460:Iodoacetamide
4-Vinylpyridine; Cysteine modification; Iodoacetamide; Mass spectrometry; N-Maleoyl amino acids; Proteomics