Monday, May 24, 2004 -- 11:55 am -- Oral Presentation

Rapid Analysis of Complex Protein Mixtures and Digests via a Very High Throughput MALDI Mass Spectrometer
Kirk S. Boraas; Noah P. Christian; James P. Reilly; Indiana University, Bloomington, IN

Introduction
Chromatographic techniques used to separate complex protein mixtures produce large numbers of fractions whose analysis would overwhelm conventional MALDI mass spectrometers. This has led to the development of a new high throughput MALDI TOF apparatus that can accommodate more than 50,000 MALDI samples and record spectra at the rate of 1 sample/sec. The system utilizes a deposition device that periodically deposits column effluent on a magnetically coated polymer tape substrate. Spooled tape and samples are placed inside a low vacuum chamber and subsequently advanced into the high vacuum MALDI TOF mass spectrometer where differential pumping isolates the bulk tape from the mass analyzer region. This system has been used to analyze chromatographically separated ribosomal proteins and bacterial protein digests.

Methods
A mixture containing 55 ribosomal proteins obtained from Caulobacter crescentus was separated via a C-4 reversed phase column, mixed with matrix and deposited on the polymer tape. The chromatographic run required 40 minutes and produced 1200 0.5µL MALDI samples. Similar experiments utilized the 2D LC/LC separation and analysis of whole cell lysates. On tape protein digests were prepared by sequentially depositing and drying trypsin, soluble protein, buffer and matrix solutions. Samples were accurately advanced through the mass analyzer's source region using coincident magnetic marks recorded on the tape at the time of deposition. Each MALDI spot was interrogated by 1000 15 µJ laser shots from a 1 kHz Nd:YLF laser steered via a high-speed laser steering system.

Preliminary Results
The mass spectrometric analysis of 1200 ribosomal protein samples required approximately 20 minutes corresponding to a thoroughput of 1 sample/sec. The analysis identified more than 40 of 55 proteins known to be present in the mixture. Magnetic markers, encoded at the time of sample deposition, enabled the accurate placement of samples within the mass analyzer's source region. Proper tape tension, a prerequisite for good mass spectrometric resolution, was monitored via a tape tension sensor. Sample analysis consisted of rapidly scanning a focused 1 kHz Nd:YLF laser beam over each MALDI spot in a logarithmic spiral using high speed laser steering mirrors. This minimized excessive sample ablation that occurs when the laser remains in one position for more than 20 milliseconds. Results also showed that a single sample may be scanned up to 15 times before significant signal loss occurs thus permitting the archiving and re-scanning of samples. Automated in-house software was responsible for data flow management and instrument control. It also permitted automatic internal and external calibration of mass spectra where external calibrants were deposited adjacent to protein samples and scanned as necessary with the laser steering system. Mass accuracy of the protein TOF mass spectrometer was determined to be +/- 1 Da. Protein LC effluent was deposited on tape every few seconds. 20-40 second wide chromatographic peaks enabled proteins to be detected in 10-20 consecutive mass spectra. In-house software was used to integrate peak areas from all 1200 mass spectra to produce ion specific chromatograms. Automated on-tape tryptic digests of proteins were found to yield expected proteolytic fragments. Conditions such as temperature, concentration and drying times were optimized to produce the most complete digestion. In combination with 2 dimensional LC/LC (ion exchange/reversed phase) chromatography, the system was used to identify individual proteins in bacterial whole cell lysates.

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