Full Extraction (In-Tube, Ethanol, Formic Acid Method)

For organisms that are not identified by the previous two techniques, a full extraction or in-tube extraction can be performed. While manufacturer recommendations may vary slightly, the general extraction methods involve the transfer of microorganisms into a tube containing MS-grade water followed by thorough vortexing. An aliquot of 100% ethanol is then added to a final concentration of 75% and is followed by a second vortex step. The sample is then pelleted through centrifugation. The supernatant is removed from the tube and equal amounts of 70% formic acid and 100% acetonitrile are added. The tube is then vortexed. The solution is then centrifuged and 1 µL of supernatant is spotted on the target plate. After drying, CHCA is added and the template is subjected to routine MALDI-TOF MS analysis.

The advantage of this technique is that the proteins are more intensely concentrated and have higher yields of acceptable identification.¹³ The disadvantage of this process is that it requires the use of harsh chemicals and includes several centrifugation steps which are more labor intensive than the previous spotting techniques.