Analyzing and distinguishing organisms such as bacterial spores by their soluble polypeptides

A method of distinguishing microorganisms or bacterial spores, comprising the steps of:

• preparing a microorganism or bacterial spore sample on a mass spectrometry sample holder;
• adding a solvent to said sample to extract selectively a limited set of proteins from the sample;
• digesting the limited set of proteins with proteolytic enzymes to produce peptide fragments;
• subjecting the peptide fragments to mass spectrometry analyses;
• comparing results of the mass spectrometry analyses of the peptide fragments with results of mass spectrometry for known bacterial spore samples;
• and
• identifying the microorganism or bacterial spore sample by matching the results of the mass spectrometry analyses of the peptide fragments with results of mass spectrometry for peptide fragments from digested small, acid-soluble proteins from at least one bacterial spore sample having a known identity, or with sequences in databases of protein and gene sequences using at least one search algorithim.

A method of identifying a Bacillus species and strain in bacterial spores, comprising the steps of:

• preparing a bacterial spore sample on a matrix-assisted laser desorption ionization time-of-flight mass spectrometry sample holder;
• adding trifluoroacetic acid to said sample to extract small, acid-soluble proteins from the sample;
• digesting the small, acid-soluble proteins with trypsin to produce peptide fragments;
• subjecting the peptide fragments to matrix-assisted laser desorption ionization time-of-flight mass spectrometry wherein any matrix is used;
• comparing results of the mass spectrometry of the peptide fragments with results of mass spectrometry for known bacterial spore samples or with sequences in global or customized databases of protein or gene sequences;
• identifying the bacterial spore sample as containing spores of at least one Bacillus species and strain by matching the results of the mass spectrometry of the peptide fragments with sequences for peptide fragments from digested small, acid-soluble proteins from at least one bacterial spore sample having a known Bacillus species and strain.

A method of preparing a library of mass spectrometry data for single cell organisms or microorganisms, comprising the steps of:

• a) preparing a sample of at least one single cell organism or microorganism;
• adding a solvent to said sample to extract small, acid-soluble proteins from the sample;
• b) digesting the small, acid-soluble proteins with proteolytic enzyme to produce peptide fragments;
• c) subjecting the peptide fragments to mass spectrometry;
• d) repeating steps a) through c) for additional organisms or microorganisms;
• e) storing data results from the mass spectrometry for each organism or microorganism in an accessible location to form a library of said data results.