Summary High‐throughput identification of proteins with the latest generation of hybrid high‐resolution mass spectrometers is opening new perspectives in microbiology. I present, here, an overview of tandem mass spectrometry technology and bioinformatics for shotgun proteomics that make 2D‐PAGE approaches obsolete. Non‐labelling quantitative approaches have become more popular than labelling techniques on most proteomic platforms because they are easier to carry out while their quantitative outcome is rather robust. Parameters for recording mass spectrometry data, however, need to be chosen carefully and statistics to assess the confidence of the results should not be neglected. Interestingly, next‐generation sequencing methodologies make any microbial model quickly amenable to proteomics, leading to the documentation of a wide range of organisms from diverse environments. Some recent discoveries made using microbial proteomics have challenged some biological dogma, such as: (i) initiation of the translation does not occur predominantly from ATG codons in some microorganisms, (ii) non‐canonical initiation codons are used to regulate the production of specific but
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