Analysis of complex proteome fractions can be accomplished through separation followed by either spot excision from a 2D gel or peak identification via liquid chromatography. Proteins of interest can then be isolated and trypsinized for peptide fragment analysis. No matter what separation technique is used in such an approach, subsequent analysis usually relies on peptide digestion for protein identification. Electrospray-mass spectrometry has been the method of choice for such identifications since the digests can be separated via a reversed-phase column and flowed directly into the ion source towards ionization. Peptide mass identification may then be performed followed by the sequencing of target peptides. The power behind such proteomic techniques relies on the combination of identification and peptide sequencing. Generated data can then be used with existing databases to produce a list of probable proteins ranked by statistical correlation to known spectra.
Electrospray-MS can yield overly complex spectra of multiply charged states that need to be deconvoluted properly in order to reach the proper peptide assignments. Additionally, it is likely that any given spot or fraction contains more than one protein and thus, these added complexities must be taken into account (increasing the chances of false/erroneous identifications). One way to circumvent the problem is by using MALDI-TOF analysis, which favors monoprotic species (one analyte, one charge state). Time-of-flight instruments do not have easy methods for sequencing peptides and thus, the trade off for sample simplicity is the loss of easy modes of peptide sequencing, markedly lowering the confidence level for protein identification. Based on demand, MassTech now offers an atmospheric pressure ion source that utilizes matrix-assisted, laser-desorption ionization followed by directed capturing within the ion trap for sensitive detection and sequencing.
Operation of this system is simple. The samples are aliquoted, either off-line or via separately sold robot, onto a MALDI-like chip (samples usually require concentrating via Zip-Tips or a similar low volume technique). The matrix is added in a manner similar to MALDI-TOF applications and there are no restrictions on matrices that can be used (most matrices used in our laboratory worked well). Once the plate is in place, the source is closed and the positioning of the plate is initialized. The laser is orthogonal to the plate, requiring fixed mirrors to direct the beam and the process is monitored by an external LCD screen ensuring alignment of the pulsed beam and the sample spot. The shots can then be either manually directed or instructed to proceed via a automated spiral pattern. All options are modifiable using the software provided.
MassTech provides its own exterior software package that works independent of the mass spectrometer’s software. This gives a multi-task feel to the package and can be automated, interfacing readily with the core instrument for easy coordination. The data results are given in the same format as any other MS spectra. The data is collected by the MS-controlling software, so the files can be saved and exported to all databases. Since the ionization technique is the only modification, ions can be subjected to any and all analyses options the ion trap has to offer.
MassTech sells the different ion sources as a complete package. There are independent units that sit near or on the ion trap, but these units have a relatively small footprint. The cost is reasonable, especially when considering the applicability of expanding ion trap capabilities to include the benefits of MALDI-ionization. The source is easily swapped out and thus, one instrument can be readily used for both MALDI- and LC/MSn, greatly improving the capabilities of the proteomics lab with a single purchase.
Ronald A. Miller
Research Biochemist
Merck Research Laboratories
Department of Alzheimer's Disease Research