For organisms that do not identify 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 (Alatoom, 2011). 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.