Abstract

BackgroundAlthough the importance of proteins of the biomineral organic matrix and their posttranslational modifications for biomineralization is generally recognized, the number of published matrix proteomes is still small. This is mostly due to the lack of comprehensive sequence databases, usually derived from genomic sequencing projects. However, in-depth mass spectrometry-based proteomic analysis, which critically depends on high-quality sequence databases, is a very fast tool to identify candidates for functional biomineral matrix proteins and their posttranslational modifications. Identification of such candidate proteins is facilitated by at least approximate quantitation of the identified proteins, because the most abundant ones may also be the most interesting candidates for further functional analysis.ResultsRe-quantification of previously identified Lottia shell matrix proteins using the intensity-based absolute quantification (iBAQ) method as implemented in the MaxQuant identification and quantitation software showed that only 57 of the 382 accepted identifications constituted 98% of the total identified matrix proteome. This group of proteins did not contain obvious intracellular proteins, such as cytoskeletal components or ribosomal proteins, invariably identified as minor components of high-throughput biomineral matrix proteomes. Fourteen of these major proteins were phosphorylated to a variable extent. All together we identified 52 phospho sites in 20 of the 382 accepted proteins with high confidence.ConclusionsWe show that iBAQ quantitation may be a useful tool to narrow down the group of functional biomineral matrix protein candidates for further research in cell biology, genetics or materials research. Knowledge of posttranslational modifications in these major proteins could be a valuable addition to previously published proteomes. This is true especially for phosphorylation, because this modification was already shown to modify mineralization processes in some instances.

Highlights

  • Phosphorylation is one of the most widespread posttranslational modifications of proteins and occurs in the organic matrix of biominerals [1,2]

  • We re-analyzed the raw-files produced previously for acid-soluble and acid-insoluble matrix prepared according to method B [17] using a combination of both databases and a subset of Uniprot containing Lottia + gigantea entries

  • To determine the approximate abundances of the identified proteins, the intensity-based absolute quantification (iBAQ) method [19] as implemented in more recent MaxQuant versions was enabled in this search

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Summary

Introduction

Phosphorylation is one of the most widespread posttranslational modifications of proteins and occurs in the organic matrix of biominerals [1,2]. The recently published genomes of biomineralizing organisms enable high-throughput mass spectrometrybased analysis of biomineral proteomes and phosphoproteomes, facilitating the fast identification of phosphoproteins and phosphorylation sites [15,16]. In-depth mass spectrometry-based proteomic analysis, which critically depends on high-quality sequence databases, is a very fast tool to identify candidates for functional biomineral matrix proteins and their posttranslational modifications. Identification of such candidate proteins is facilitated by at least approximate quantitation of the identified proteins, because the most abundant ones may be the most interesting candidates for further functional analysis

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