Orbi is running second set of Scott’s samples; the first set has been analyzed and the data processed. Next in the queue are Eric’s and Josh’s samples, after which the instrument will be shut down for the holidays.
MALDI will also be shut down tomorrow to avoid potential problems (based on the last year’s holidays experience).
I feel terrible about the imminent (and anxiously anticipated) complete destruction of this work of art. Tonight. That’s when it is going to happen. Sorry, Marlena.
Today is Monday. Tomorrow is safety inspection. I am extracting 48 gel slices.
On the bright side, Yuanjun’s data is crunching and should be ready by tomorrow, and the lights on all our boxes are green!
Orbi is running Ninad’s IPs. Eric’s samples are next. Scott’s gel lanes will be ready for proteolysis by tomorrow morning. We have heating, cooling, and electricity, all at the same time!
MALDI is experiencing severe deficit of your attention.
We are two weeks away from the winter holiday break (12/22 – 01/04), during which Orbi will be down. This morning, I received 48 samples for Orbi; so counting the 24 samples currently running, plus 6 more waiting, the Orbi queue is completely full for the 2 weeks before the break. I ask you to keep your samples safe in your refrigerators or freezers until we return in January.
MALDI currently doesn’t have a queue.
The MALDI Biotyper identifies microorganisms by analyzing their intrinsic proteins using mass spectrometry.
An individual colony from an overnight culture grown on an agar plate is picked using a wooden toothpick and transferred to a MALDI target. This sample is allowed to dry, and a microliter of a matrix solution is added to the sample. The organic solvent in the matrix solution extracts proteins from the microorganisms. The extracted proteins are mainly ribosomal proteins, present in high concentrations. Once the matrix has crystallized, sample preparation is complete and the samples are ready for analysis. For some microorganisms, it is necessary to perform a more extensive extraction, which adds another 10-15 minutes to the sample preparation procedure.
Samples are ready to be analyzed. (MSP 96 target polished steel; Bruker Part No: 8280800)
In our facility, the analyses are carried out on the Bruker Ultraflextreme MALDI TOF/TOF mass spectrometer. Each MALDI mass spectrum is a species-specific molecular fingerprint, a mass and intensity distribution of peaks corresponding to mainly ribosomal proteins.
Microorganisms are identified in real time as soon as data are collected from each individual target position.
Identification of an unknown microorganism is based on comparing the experimental molecular fingerprint with 6903 fingerprints in the Biotyper library and assigning a score which represents the probability that the match is correct.
Off-line classification software provides additional options for working with data such as creating new library entries, re-organizing libraries, viewing mass spectra, or assessing quality of match.
The algorithm used to compare patterns computes three separate values for three fundamental characteristics of the sample spectra and the reference spectra: the number of signals in the reference spectrum that have a closely matching partner in the unknown spectrum; the number of signals in the unknown spectrum that have a closely matching partner in the reference spectrum; and the intensity symmetry of the matching pairs. The maximum obtainable score is 3, and the score values greater or equal to 2 are considered as a probable identification.
Each mass spectrum is converted into a histogram which represents the mass and intensity ‘fingerprint’.
Biotyper software allows building spectral libraries of any type of samples as long as the samples are amenable to MALDI-TOF MS and produce unique MS fingerprints. If you are interested in learning more about MALDI MS and the Biotyper, please contact Tatiana.
The acquisition of MALDI Biotyper software was possible through collaboration with Dr. Bhushan M. Jayarao, Director of Penn State Animal Diagnostic Laboratory, Professor of Veterinary and Biomedical Sciences.
Featured image credit: Rocky Mountain Laboratories, NIAID, NIH
Scanning electron micrograph of Escherichia coli, grown in culture and adhered to a coverslip
We had a power interruption today to allow electricians repair a faulty circuit breaker. All mass spectrometers were down, and now we are getting back to operating conditions. This will cause some delays in your sample analyses.
I should be able to resume running Yuanjun’s samples on Monday, after Orbi gets stable temperature so I can calibrate it.
MALDI is ready.
LCT is pumping down.
QTrap is pumping down.
Today marks my four years at Penn State.
Orbi is running John’s and Brian’s samples. Next are Ninad’s IP samples and Yuanjun’s set of 32 samples. This means Orbi will be busy until the end of this week, unless someone cuts the power again.
MALDI is bored.