Many mitochondrial proteins are synthesized with N-terminal presequences that are removed by specific peptidases. The N-termini of the mature proteins and thus peptidase cleavage sites have only been determined for a small fraction of mitochondrial proteins and yielded a controversial situation for the cleavage site specificity of the major mitochondrial processing peptidase (MPP). We report a global analysis of the N-proteome of yeast mitochondria, revealing the N-termini of 615 different proteins. Significantly more proteins than predicted contained cleavable presequences. We identified the intermediate cleaving peptidase Icp55, which removes an amino acid from a characteristic set of MPP-generated N-termini, solving the controversial situation of MPP specificity and suggesting that Icp55 converts instable intermediates into stable proteins. Our results suggest that Icp55 is critical for stabilization of the mitochondrial proteome and illustrate how the N-proteome can serve as rich source for a systematic analysis of mitochondrial protein targeting, cleavage and turnover.

Stable isotope labelling in combination with mass spectrometry has emerged as a powerful tool to identify and relatively quantify thousands of proteins within complex protein mixtures. Here we describe a novel method, termed isotope-coded protein label (ICPL), which is capable of high-throughput quantitative proteome profiling on a global scale. Since ICPL is based on stable isotope tagging at the frequent free amino groups of isolated intact proteins, it is applicable to any protein sample, including extracts from tissues or body fluids, and compatible to all separation methods currently employed in proteome studies. The method showed highly accurate and reproducible quantification of proteins and yielded high sequence coverage, indispensable for the detection of post-translational modifications and protein isoforms. The efficiency (e.g. accuracy, dynamic range, sensitivity, speed) of the approach is demonstrated by comparative analysis of two differentially spiked proteomes.

We describe a method for correlating the immunoglobulin (Ig) proteomes with the B cell transcriptomes in human fluid and tissue samples, using multiple sclerosis as a paradigm. Oligoclonal Ig bands and elevated numbers of clonally expanded B cells in the cerebrospinal fluid (CSF) are diagnostic hallmarks of multiple sclerosis. Here we compared the Ig transcriptomes of B cells with the corresponding Ig proteomes in CSF samples from four subjects with multiple sclerosis. We created individual Ig transcriptome databases that contained the subject-specific mutations introduced by V(D)J recombination and somatic hypermutation and then searched the CSF for corresponding characteristic peptides by mass spectrometry. In each sample, the Ig transcriptomes and proteomes strongly overlapped, showing that CSF B cells indeed produce the oligoclonal Ig bands. This approach can be applied to other organ-specific diagnostic fluid or tissue samples to compare the Ig transcripts of local B cells with the corresponding antibody proteomes of individual subjects.

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We have utilized a T7 polymerase/promoter system for the high‐level incorporation of methionine analogs with suitable labels for structural research (X‐ray and NMR studies) on recombinant annexin V produced in Escherichia coli. Here, we describe, to our knowledge, the first biosynthetic high‐level substitution of methionine by 2‐aminohexanoic acid (norleucine), ethionine and telluromethionine in a protein. The replacement has been confirmed by electrospray mass spectroscopy, amino acid analysis and X‐ray structural analysis. Conditions for expression were optimized concerning the frequency of appearance of revertants, high‐level replacement and maximal protein yield. For the incorporation of norleucine and ethionine, E. coli B834 (DE3)(hsd metB), which is auxotrophic for methionine, was grown under methionine‐limited conditions with an excess of the analog in the culture medium, and the expression of protein under the control of the T7 promoter was induced after the methionine supply had been exhausted. The factor limiting the high‐level incorporation of telluromethionine into protein is its sensitivity towards oxidation. To overcome this problem, bacteria were grown with a limited amount of methionine, harvested after its exhaustion and resuspended in fresh media without methionine; telluromethionine was added and protein synthesis induced. Under these conditions, significant amounts of protein can be expressed before telluromethionine has been completely degraded (within hours). Biosynthetic incorporation of heavy atoms such as tellurium into recombinant proteins can accelerate the process of obtaining heavy‐atom derivatives suitable for X‐ray structural analysis, supplementing the traditional trial‐and‐error preparation of heavy‐atom derivatives for the method of multiple isomorphous replacement. Furthermore, the successful high‐level incorporation of amino acid analogs can provide single‐atom mutations for the detailed study of the structure and function of proteins.