Thomas Wenzel

Immuno-MALDI-TOF MS: new perspectives for clinical applications of mass spectrometry.

The discovery of novel biomarkers by means of advanced detection tools based on proteomic analysis technologies necessitates the development of improved diagnostic methods for application in clinical routine. On the basis of three different application examples, this review presents the limitations of conventional routine diagnostic assays and illustrates the advantages of immunoaffinity enrichment combined with MALDI‐TOF MS. Applying this approach increases the specificity of the analysis supporting a better diagnostic recognition, sensitivity, and differentiation of certain diseases. The use of MALDI‐TOF MS as detection method facilitates the identification of modified peptides and proteins providing additional information. Further, employing respective internal standard peptides allows for relative and absolute quantitation which is mandatory in the clinical context. Although MALDI‐TOF MS is not yet established for clinical routine diagnostics this technology has a high potential for improvement of clinical diagnostics and monitoring therapeutic efficacy.

Genosnip: SNP Genotyping by MALDI-TOF MS Using Photocleavable Oligonucleotides

Abstract A photocleavable o-nitrobenzyl CE phosphoramidite building-block was synthesised and incorporated within oligonucleotides. After allele-specific primer extension, desalting was performed using genostrep purification plates. Release of the SNP information containing part through photocleavage created shortened molecules that are easily accessible for MALDI-TOF analysis. Additionally, incorporation of mass modified nucleosides enables flexible design of multiplex genotyping.

Synthesis of 1-Deazaadenosine Analogues of (2′→5′) ApApA

Abstract Synthesis of (2′ → 5′)ApApA analogues containing 1-deazaadenosine at different positions is described (32–34). The approach used the phosphotrieer methodology in solution and utilized 3′-O-benzoylated derivatives of the N6-protected 5′-O-monomethoxytrityl-1-deazaadenosine as starting material. Dedicated to Prof. Y. Mizuno on the occasion of his 75th birthday

NOVEL PROPARGYLAMINE-LINKED NUCLEOSIDES FOR HIGH THROUGHPUT SNP GENOTYPING BY MALDI-TOF MS

The synthesis of positively charged and mass tagged nucleosides containing a quaternary ammonium functionality within the penultimate position of a primer is described. Neutralization of the sugar/thiophosphate backbone by alkylation increases the detection sensitivity in the mass spectrometric analysis by a factor of at least 100. The variable introduction of these novel compounds within the extension primers enables flexible design of multiplex genotyping reactions.

Hoogsteen-Duplex DNA: Synthesis and Base Pairing of Oligodeoxynucleotides Containing 1-Deaza-2′-deoxyadenosine

Abstract Solid-phase synthesis of oligonucleotides containing 1-deazaadenine was carried out employing phosphonate and phosphoramidite chemistry. Hoogsteen base pairing was established for the duplex d(c1A20)·d(T20).

Strategies for SNP genotyping by mass spectrometry

Abstract Single nucleotide polymorphisms (SNPs) represent the most frequently found sequence variations in the human genome. Thus, SNPs are thought to be useful markers for the identification of genetic factors associated with complex diseases, differential pharmacogenetic response as well as for the analysis of forensic samples. Here, we describe the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for the analysis of SNPs associated with a risk for common complex diseases. For highly efficient monitoring of a set of relevant SNP markers, the mass spectrometric analysis has been integrated into an overall process including the set-up of allele specific reactions, sample preparation and automated allele calling.