11 research outputs found
Mixed-Bed Ion Exchange Chromatography Employing a Salt-Free pH Gradient for Improved Sensitivity and Compatibility in MudPIT
In
proteomics, comprehensive analysis of peptides mixtures necessitates
multiple dimensions of separation prior to mass spectrometry analysis
to reduce sample complexity and increase the dynamic range of analysis.
The main goal of this work was to improve the performance of (online)
multidimensional protein identification technology (MudPIT) in terms
of sensitivity, compatibility and recovery. The method employs weak
anion and strong cation mixed-bed ion exchange chromatography (ACE)
in the first separation dimension and reversed phase chromatography
(RP) in the second separation dimension (Motoyama et.al. <i>Anal.
Chem</i> <b>2007</b>, <i>79</i>, 3623â34.).
We demonstrated that the chromatographic behavior of peptides in ACE
chromatography depends on both the WAX/SCX mixing ratio as the ionic
strength of the mobile phase system. This property allowed us to replace
the conventional salt gradient by a (discontinuous) salt-free, pH
gradient. First dimensional separation of peptides was accomplished
with mixtures of aqueous formic acid and dimethylsulfoxide with increasing
concentrations. The overall performance of this mobile phase system
was found comparable to ammonium acetate buffers in application to
ACE chromatography, but clearly outperformed strong cation exchange
for use in first dimensional peptide separation. The dramatically
improved compatibility between (salt-free) ion exchange chromatography
and reversed phase chromatographyâmass spectrometry allowed
us to downscale the dimensions of the RP analytical column down to
25 ÎŒm i.d. for an additional 2- to 3-fold improvement in performance
compared to current technology. The achieved levels of sensitivity,
orthogonality, and compatibility demonstrates the potential of salt-free
ACE MudPIT for the ultrasensitive, multidimensional analysis of very
modest amounts of sample material
Mixed-Bed Ion Exchange Chromatography Employing a Salt-Free pH Gradient for Improved Sensitivity and Compatibility in MudPIT
In
proteomics, comprehensive analysis of peptides mixtures necessitates
multiple dimensions of separation prior to mass spectrometry analysis
to reduce sample complexity and increase the dynamic range of analysis.
The main goal of this work was to improve the performance of (online)
multidimensional protein identification technology (MudPIT) in terms
of sensitivity, compatibility and recovery. The method employs weak
anion and strong cation mixed-bed ion exchange chromatography (ACE)
in the first separation dimension and reversed phase chromatography
(RP) in the second separation dimension (Motoyama et.al. <i>Anal.
Chem</i> <b>2007</b>, <i>79</i>, 3623â34.).
We demonstrated that the chromatographic behavior of peptides in ACE
chromatography depends on both the WAX/SCX mixing ratio as the ionic
strength of the mobile phase system. This property allowed us to replace
the conventional salt gradient by a (discontinuous) salt-free, pH
gradient. First dimensional separation of peptides was accomplished
with mixtures of aqueous formic acid and dimethylsulfoxide with increasing
concentrations. The overall performance of this mobile phase system
was found comparable to ammonium acetate buffers in application to
ACE chromatography, but clearly outperformed strong cation exchange
for use in first dimensional peptide separation. The dramatically
improved compatibility between (salt-free) ion exchange chromatography
and reversed phase chromatographyâmass spectrometry allowed
us to downscale the dimensions of the RP analytical column down to
25 ÎŒm i.d. for an additional 2- to 3-fold improvement in performance
compared to current technology. The achieved levels of sensitivity,
orthogonality, and compatibility demonstrates the potential of salt-free
ACE MudPIT for the ultrasensitive, multidimensional analysis of very
modest amounts of sample material
Arginine (Di)methylated Human Leukocyte Antigen Class I Peptides Are Favorably Presented by HLA-B*07.
Alterations in protein post-translational modification (PTM) are recognized hallmarks of diseases. These modifications potentially provide a unique source of disease-related human leukocyte antigen (HLA) class I-presented peptides that can elicit specific immune responses. While phosphorylated HLA peptides have already received attention, arginine methylated HLA class I peptide presentation has not been characterized in detail. In a human B-cell line we detected 149 HLA class I peptides harboring mono- and/or dimethylated arginine residues by mass spectrometry. A striking preference was observed in the presentation of arginine (di)methylated peptides for HLA-B*07 molecules, likely because the binding motifs of this allele resemble consensus sequences recognized by arginine methyl-transferases. Moreover, HLA-B*07-bound peptides preferentially harbored dimethylated groups at the P3 position, thus consecutively to the proline anchor residue. Such a proline-arginine sequence has been associated with the arginine methyl-transferases CARM1 and PRMT5. Making use of the specific neutral losses in fragmentation spectra, we found most of the peptides to be asymmetrically dimethylated, most likely by CARM1. These data expand our knowledge of the processing and presentation of arginine (di)methylated HLA class I peptides and demonstrate that these types of modified peptides can be presented for recognition by T-cells. HLA class I peptides with mono- and dimethylated arginine residues may therefore offer a novel target for immunotherapy
Arginine (Di)methylated Human Leukocyte Antigen Class I Peptides Are Favorably Presented by HLA-B*07
Alterations in protein post-translational modification (PTM) are recognized hallmarks of diseases. These modifications potentially provide a unique source of disease-related human leukocyte antigen (HLA) class I-presented peptides that can elicit specific immune responses. While phosphorylated HLA peptides have already received attention, arginine methylated HLA class I peptide presentation has not been characterized in detail. In a human B-cell line we detected 149 HLA class I peptides harboring mono- and/or dimethylated arginine residues by mass spectrometry. A striking preference was observed in the presentation of arginine (di)methylated peptides for HLA-B*07 molecules, likely because the binding motifs of this allele resemble consensus sequences recognized by arginine methyl-transferases. Moreover, HLA-B*07-bound peptides preferentially harbored dimethylated groups at the P3 position, thus consecutively to the proline anchor residue. Such a proline-arginine sequence has been associated with the arginine methyl-transferases CARM1 and PRMT5. Making use of the specific neutral losses in fragmentation spectra, we found most of the peptides to be asymmetrically dimethylated, most likely by CARM1. These data expand our knowledge of the processing and presentation of arginine (di)methylated HLA class I peptides and demonstrate that these types of modified peptides can be presented for recognition by T-cells. HLA class I peptides with mono- and dimethylated arginine residues may therefore offer a novel target for immunotherapy
Novel identified aluminum hydroxide-induced pathways proof monocyte activation and pro-inflammatory preparedness.
Aluminum-based adjuvants are the most widely used adjuvants in human vaccines. A comprehensive understanding of the mechanism of action of aluminum adjuvants at the molecular level, however, is still elusive. Here, we unravel the effects of aluminum hydroxide Al(OH)3 by a systems-wide analysis of the Al(OH)3-induced monocyte response. Cell response analysis by cytokine release was combined with (targeted) transcriptome and full proteome analysis. Results from this comprehensive study revealed two novel pathways to become activated upon monocyte stimulation with Al(OH)3: the first pathway was IFNÎČ signaling possibly induced by DAMP sensing pathways like TLR or NOD1 activation, and second the HLA class I antigen processing and presentation pathway. Furthermore, known mechanisms of the adjuvant activity of Al(OH)3 were elucidated in more detail such as inflammasome and complement activation, homeostasis and HLA-class II upregulation, possibly related to increased IFNÎł gene expression. Altogether, our study revealed which immunological pathways are activated upon stimulation of monocytes with Al(OH)3, refining our knowledge on the adjuvant effect of Al(OH)3 in primary monocytes
Extended O-GlcNAc on HLA Class-I-Bound Peptides
We report unexpected mass spectrometric observations of glycosylated human leukocyte antigen (HLA) class I-bound peptides. Complemented by molecular modeling, in vitro enzymatic assays, and oxonium ion patterns, we propose that the observed O-linked glycans carrying up to five monosaccharides are extended O-GlcNAc's rather than GalNAc-initiated O-glycans. A cytosolic O-GlcNAc modification is normally terminal and does not extend to produce a polysaccharide, but O-GlcNAc on an HLA peptide presents a special case because the loaded HLA class I complex traffics through the endoplasmic reticulum and Golgi apparatus on its way to the cell membrane and is hence exposed to glycosyltransferases. We also report for the first time natural HLA class I presentation of O- and N-linked glycopeptides derived from membrane proteins. HLA class I peptides with centrally located oligosaccharides have been shown to be immunogenic and may thus be important targets for immune surveillance
Extended O-GlcNAc on HLA Class-I-Bound Peptides
We report unexpected mass spectrometric observations of glycosylated human leukocyte antigen (HLA) class I-bound peptides. Complemented by molecular modeling, in vitro enzymatic assays, and oxonium ion patterns, we propose that the observed O-linked glycans carrying up to five monosaccharides are extended O-GlcNAc's rather than GalNAc-initiated O-glycans. A cytosolic O-GlcNAc modification is normally terminal and does not extend to produce a polysaccharide, but O-GlcNAc on an HLA peptide presents a special case because the loaded HLA class I complex traffics through the endoplasmic reticulum and Golgi apparatus on its way to the cell membrane and is hence exposed to glycosyltransferases. We also report for the first time natural HLA class I presentation of O- and N-linked glycopeptides derived from membrane proteins. HLA class I peptides with centrally located oligosaccharides have been shown to be immunogenic and may thus be important targets for immune surveillance
Quantitative Proteomics Reveals Distinct Differences in the Protein Content of Outer Membrane Vesicle Vaccines
At
present, only vaccines containing outer membrane vesicles (OMV)
have successfully stopped <i>Neisseria meningitidis</i> serogroup
B epidemics. These vaccines however require detergent-extraction to
remove endotoxin, which changes immunogenicity and causes production
difficulties. To investigate this in more detail, the protein content
of detergent-extracted OMV is compared with two detergent-free alternatives.
A novel proteomics strategy has been developed that allows quantitative
analysis of many biological replicates despite inherent multiplex
restrictions of dimethyl labeling. This enables robust statistical
analysis of relative protein abundance. The comparison with detergent-extracted
OMV reveales that detergent-free OMV are enriched with membrane (lipo)Âproteins
and contain less cytoplasmic proteins due to a milder purification
process. These distinct protein profiles are substantiated with serum
blot proteomics, confirming enrichment with immunogenic proteins in
both detergent-free alternatives. Therefore, the immunogenic protein
content of OMV vaccines depends at least partially on the purification
process. This study demonstrates that detergent-free OMV have a preferred
composition
Extended OâGlcNAc on HLA Class-I-Bound Peptides
We report unexpected mass spectrometric
observations of glycosylated
human leukocyte antigen (HLA) class I-bound peptides. Complemented
by molecular modeling, <i>in vitro</i> enzymatic assays,
and oxonium ion patterns, we propose that the observed O-linked glycans
carrying up to five monosaccharides are extended O-GlcNAcâs
rather than GalNAc-initiated O-glycans. A cytosolic O-GlcNAc modification
is normally terminal and does not extend to produce a polysaccharide,
but O-GlcNAc on an HLA peptide presents a special case because the
loaded HLA class I complex traffics through the endoplasmic reticulum
and Golgi apparatus on its way to the cell membrane and is hence exposed
to glycosyltransferases. We also report for the first time natural
HLA class I presentation of O- and N-linked glycopeptides derived
from membrane proteins. HLA class I peptides with centrally located
oligosaccharides have been shown to be immunogenic and may thus be
important targets for immune surveillance