An antibody-based biomarker discovery method by mass spectrometry sequencing of complementarity determining regions

Autoantibodies are increasingly used as biomarkers in the detection of autoimmune disorders and cancer. Disease specific antibodies are generally detected by their binding to specific antigens. As an alternative approach, we propose to identify specific complementarity determining regions (CDR) of IgG that relate to an autoimmune disorder or cancer instead of the specific antigen(s). In this manuscript, we tested the technical feasibility to detect and identify CDRs of specific antibodies by mass spectrometry. We used a commercial pooled IgG preparation as well as purified serum IgG fractions that were spiked with different amounts of a fully human monoclonal antibody (adalimumab). These samples were enzymatically digested and analyzed by nanoLC Orbitrap mass spectrometry. In these samples, we were able to identify peptides derived from the CDRs of adalimumab. These peptides could be detected at an amount of 110 attomole, 5 orders of magnitude lower than the total IgG concentration in these samples. Using higher energy collision induced dissociation (HCD) fragmentation and subsequent de novo sequencing, we could successfully identify 50% of the detectable CDR peptides of adalimumab. In addition, we demonstrated that an affinity purification with anti-dinitrophenol (DNP) monoclonal antibody enhanced anti-DNP derived CDR detection in a serum IgG background. In conclusion, specific CDR peptides could be detected and sequenced at relatively low levels (attomole-femtomole range) which should allow the detection of clinically relevant CDR peptides in patient samples

Perturbation of the yeast N-acetyltransferase NatB induces elevation of protein phosphorylation levels

<p>Abstract</p> <p>Background</p> <p>The addition of an acetyl group to protein N-termini is a widespread co-translational modification. NatB is one of the main N-acetyltransferases that targets a subset of proteins possessing an N-terminal methionine, but so far only a handful of substrates have been reported. Using a yeast <it>nat3Δ </it>strain, deficient for the catalytic subunit of NatB, we employed a quantitative proteomics strategy to identify NatB substrates and to characterize downstream effects in <it>nat3Δ</it>.</p> <p>Results</p> <p>Comparing by proteomics WT and <it>nat3Δ </it>strains, using metabolic ¹⁵N isotope labeling, we confidently identified 59 NatB substrates, out of a total of 756 detected acetylated protein N-termini. We acquired in-depth proteome wide measurements of expression levels of about 2580 proteins. Most remarkably, NatB deletion led to a very significant change in protein phosphorylation.</p> <p>Conclusions</p> <p>Protein expression levels change only marginally in between WT and <it>nat3Δ</it>. A comparison of the detected NatB substrates with their orthologous revealed remarkably little conservation throughout the phylogenetic tree. We further present evidence of post-translational N-acetylation on protein variants at non-annotated N-termini. Moreover, analysis of downstream effects in <it>nat3Δ </it>revealed elevated protein phosphorylation levels whereby the kinase Snf1p is likely a key element in this process.</p

Imaging Mass Spectrometry: Hype or Hope?

Imaging mass spectrometry is currently receiving a significant amount of attention in the mass spectrometric community. It offers the potential of direct examination of biomolecular patterns from cells and tissue. This makes it a seemingly ideal tool for biomedical diagnostics and molecular histology. It is able to generate beautiful molecular images from a large variety of surfaces, ranging from cancer tissue sections to polished cross sections from old-master paintings. What are the parameters that define and control the implications, challenges, opportunities, and (im)possibilities associated with the application of imaging MS to biomedical tissue studies. Is this just another technological hype or does it really offer the hope to gain new insights in molecular processes in living tissue? In this critical insight this question is addressed through the discussion of a number of aspects of MS imaging technology and sample preparation that strongly determine the outcome of imaging MS experiments

The Leukemia-Associated Mllt10/Af10-Dot1l Are Tcf4/β-Catenin Coactivators Essential for Intestinal Homeostasis

The leukemia-associated Mllt10/Af10 and its partner the histone methyltransferase Dot1l are identified as Tcf4/β-catenin co-activators and shown to be essential for Wnt-driven endogenous gene expression, intestinal development and homeostasis

Lys-N: A versatile enzyme for proteomics

To overcome the difficulties of analyzing proteins in highly complex samples an improvement in proteomics strategies is needed. The combination of multiple proteases, peptide separation and fragmentation techniques may reduce sample complexity and improve the analysis of different sub-groups of peptides, including low abundant proteins and peptides. In this thesis, I introduce a relatively new protease in proteomics workflows and demonstrate the strength of combining its proteolytic peptides with multi-dimensional separation techniques and different fragmentation techniques, to decrease sample complexity and to improve sample identification. In chapter 2, we evaluate the fragmentation pattern observed for peptides generated by the metalloendopeptidase Lys-N using electron transfer dissociation (ETD). The enzyme Lys-N generates peptides with a lysine residue at the N-terminal. We show that ETD sequencing of BSA generated peptides with an N-terminal lysine, and no other basic residue in the sequence, result in spectra dominated by c-type fragment ions. To confirm the result, fragment ion statistics were increased by analyzing Lys-N generated peptides from a cell lysate. Additionally, we show that these doubly charged Lys-N peptides containing a single lysine at the N-terminal can be selectively enriched for by using low-pH strong cation exchange (SCX). In chapter 3, we further evaluate the SCX based fractionation of peptides generated from the metalloendopeptidase Lys-N. Here, we interestingly show that it is possible to obtain fractionation profiles where different subgroups of Lys-N generated peptides such as, acetylated N-terminal peptides, singly phosphorylated peptides, peptides with a single basic residue and peptides with multiple basic residues can be separated. We demonstrate that the combination of Lys-N digestion, low-pH SCX and reversed phase (RP) separation, with CID and ETD induced fragmentation, is a powerful approach for global proteome and phosphoproteome analysis. In chapter 4, the metalloendopeptidase was explored for its use in MALDI-MS/MS proteomics applications. Lys-N generated peptides from a BSA digest were analyzed by MALDI-MS/MS, which resulted in simple and straightforward CID spectra, containing complete b-ion series. Statistical analysis was again performed to confirm the results where a cell lysate was digested to obtain a higher number of doubly charged Lys-N peptides. Last, it was found that the simple straightforward MALDI CID spectra can be used to facilitate de novo sequencing. In chapter 5, the proteolytic performance of the metalloendopeptidase Lys-N was evaluated. As a model system BSA was used to validate the performance of Lys-N when using a number of classical proteomics sample handling conditions. We demonstrate that Lys-N has many interesting and useful characteristics as it was found to be highly thermo-stable and to have a high tolerance towards certain denaturing agents, such as urea and acetonitrile. Furthermore, it was found that by increasing the digestion temperature a decrease in incubation time could be achieved. Additionally, we demonstrate that Lys-N is able to cleave adjacent to single-methylated lysines and partially adjacent to di-methylated lysines, which may be useful when analyzing naturally occurring post-translational modification

Evaluation of metalloendopeptidase Lys-N protease performance under different sample handling conditions.

Trypsin, the most widely used enzyme in proteomics, has a few caveats as it does not perform well under certain harsh sample handling conditions and creates relatively short peptides less amenable to, for instance, electron transfer dissociation. There is, thus, room for improvement using alternative proteases. Here, we evaluate the performance of such an alternative protease, the metalloendopeptidase Lys-N, in sample preparation for proteomic analyses under various experimental conditions. The experimental parameters we evaluated were protein-to-protease ratio, incubation time, temperature, and several concentrations of denaturing modifiers often used in proteomics sample handling. Our data reveal that Lys-N is still very efficient under some very harsh (denaturing) conditions (e.g., 8 M urea, 80% acetonitrile) and at temperatures as low as 4 degrees C and up to 80 degrees C but severely hampered by guanidine hydrochloride and methanol. These rather unique features make Lys-N a good candidate for a variety of applications, such as membrane proteomics and possibly H/D exchange mass spectrometry. Additionally, we show that Lys-N is capable of, in contrast to trypsin or Lys-C, cleaving adjacent to mono- and dimethylated lysines, making it a good candidate for targeted epigenetic analysis of for instance histones

Exploring new proteome space: combining Lys-N proteolytic digestion and strong cation exchange (SCX) separation in peptide-centric MS-driven proteomics.

The current advances in mass spectrometry technology have led to the possibility of analyzing more complex biological samples such as entire proteomes. Here, we describe a new and powerful methodology that combines the use of the metalloendopeptidase Lys-N and strong cation exchange with mass spectrometric analysis. The approach described here allows one to separate peptides with different functional groups. The peptides we are able to isolate are N-terminal peptides, phosphorylated peptides with a single lysine, peptides with a single basic residue (lysine), and peptides with multiply basic residues. When this separation strategy is combined with tandem mass spectrometry that involves both collision-induced dissociation and electron transfer dissociation, one can achieve an optimal targeted strategy for proteome analysis