Shimadzu demonstrated the ability of its Perfinity workstation, which it describes as an integrated online protein sample-preparation platform combining the selectivity of immunoaffinity, the speed of online digestion, and the resolving power of LC/MS.
“Performing these steps off-line requires as little as 8 hours, but frequently greater than 24 hours. With the Perfinity workstation the full process can be completed in as little as 30 minutes,” said Rachel Lieberman, Ph.D., applications scientist.
Key to performance is use of the Perfinity immobilized trypsin column where the equivalent of a 24-hour digestion can be obtained in 60 seconds.
“The application of mass spectrometry to clinical samples depends upon mass spectrometry outperforming ELISA tests,” noted Dr. Lieberman. “The detection of isoforms is one such example. Hemoglobin is an often-sited example of where a single amino acid variation is of significant clinical relevance. As such the hemoglobin variant study was performed as a proof of concept for the demonstration of the system.”
While cycle times can be as little as seven minutes for certain applications, in cases where extremely large numbers of samples (≥ 200) must be run in less than 24 hours the end user is better off using a plate-based technique, said Dr. Lieberman. Furthermore, the current configuration of the Perfinity workstation operates at analytical flow rates and it is not suited for lower abundant biomarkers.
Having cut digestion time down to as little as one minute the pretreatment (reduction and alkylation) steps comprise the new bottleneck.
“Our current goal is to dramatically reduce the impact of these steps. Improvements that enable increased sensitivity and higher throughput are also in progress,” pointed out Dr. Lieberman.
Used properly, statistical DOE (design of experiments) techniques can be more efficient and produce better results than OFAT (change one process factor at a time) in many applications. Tecan discussed a study in which DOE, automation of sample preparation, and multiparameter LC-MS/MS analysis enabled efficient and reproducible optimization of sample prep and yield for immunosuppressant drugs simultaneously.
In the study, a half factorial design was employed with five parameters at two levels, where a total of 15 parameters were screened in three sessions (five parameters each). The critical factors identified proved to work well (e.g., increase of signal in this study).
Although powerful, manual execution of DOE series is error-prone and tedious. To mitigate this problem, Tecan used an automated liquid handler (Freedom EVO®) driven by flexible software (Freedom EVOware®) that can pipette according to dedicated scripts using input for variables from Excel tables.
“In such a table, the concentrations defined in the DOE software can be converted to liquid volumes to be pipetted, if appropriate stock solutions representing all factors are defined,” explained Roland Geyer, Ph.D., innovation manager, Tecan.
Another key piece of technology used is Tecan’s AC extraction plate, still in development, which facilitates sample cleanup by a modified type of Liquid-Liquid extraction.
“It consists of a deep-well plate with wells coated with an immobilized liquid used as the extraction substrate. The coated-well surface as such enables simplification of the entire sample-preparation process. The procedure works without any protein precipitation, filtration, centrifugation or solvent evaporation, hence accelerating and streamlining the complete sample-preparation workflow,” said Dr. Geyer.
New Interface for LC-MALDI
Development of MALDI-TOF/TOF instruments with acquisition rates up to 1,000 Hz, has made LC-MALDI-MS a viable alternative to LC-ESI-MS for many proteomic and peptidomic applications. Despite this, LC-MALDI-MS is not nearly as widely used, said Johan Gobom, Ph.D., associate professor, University of Gothenburg, Institute of Neuroscience and Physiology.
“We believe an important reason for this is the robustness and ease of use of the LC-MALDI interface,” he said.
Dr. Gobom and colleagues have developed a LC-MALDI interface based on solenoid-valve microdispensing jets for contact-free deposition of analyte and matrix on the MALDI sample plate. This overcomes some of the problems associated with the currently available LC-MALDI interfaces.
Example: use of syringe pumps to tee in the matrix solution, and fractionating by contact deposition are prone to interruptions caused by matrix crystallization in the interface and low tolerance to deviations in the x-y-z calibration of the fraction collector.
Contact-free sample fractionation for LC-MALDI is likely to be adopted quickly and widely among LC-MALDI users, said Dr. Gobom. The solenoid-valve based technology used in this work is inexpensive, and the cost of the dispensing robot is similar to that of an ordinary LC-MALDI spotter.
Plus, the new interface increases the walk-away time of LC-MALDI; a technician can load a sample tray on the LC and leave the system to fractionate the samples unattended for several days.
The described LC-MALDI interface was developed in a collaborative project between University of Gothenburg, Sweden and M2 Automation. A LC-MALDI fractionation robot based on the developed technology has been commercialized by M2 Automation.