Tuesday, May 25, 2004 -- Poster 453

Capillary LC/MALDI/TOFTOF MS Analysis of Complex Biological Samples
William R. Alley; Tony J. Tegeler; Yehia S. Mechref; Milos V. Novotny; Indiana University, Bloomington, IN

Introduction
Until recently, proteomic analysis of complex biological samples has been typically conducted using electrospray ionization. However, recent developments in our laboratory as well as others have made it possible to collect capillary LC effluents on MALDI plates for MALDI-based MS analysis. MALDI/TOFTOF MS analysis has several advantages including the elimination of on-the-fly precursor ion selection, thus permitting the performance of the MSMS analysis on the ions at the apex of the chromatographic peak rather than the edges. This would substantially improve the quality of the MSMS data acquired and consequently allow better protein identification. Moreover, deposition of an LC run on MALDI plate(s), which could be stored for a fairly long time, permits revisiting of analysis if deemed necessary.

Methods
The biological samples analyzed were reduced, alkylated, and tryptically digested prior to LC/MALDI/TOFTOF MS analysis. Samples were separated either by reversed-phase capillary LC or 2D LC. A 15-cm long capillary (100 µm i.d.) packed with C18 resin (3 µm, 120 Å) was used for the reversed-phase separation, while another 15-cm long capillary packed with 10 cm of C18 resin followed by strong cation-exchange resin was used for the 2D separation. The LC effluent was mixed with the MALDI matrix and deposited on MALDI plates using our MALDI deposition device. Our system is capable of depositing an entire run on a single plate (a single salt step) or over multiple plates (the liver and brain samples).

Preliminary Results
More peptides with better MSMS quality are observed due to the decoupling of LC from MS analysis. Consequently, more proteins are identified with higher ion scores. The total proteomes extracted from the livers or brains of alcohol-preferring rats were analyzed with a three-hour gradient. MS and tandem MS analysis of these samples allowed the identification of ca. 400 proteins. The posttranslational modifications of some of these proteins were also assigned using the tandem MS data in conjunction with database searching using MASCOT. Due to the extreme complexity of human blood serum, single-dimensional chromatography did not provide adequate peak capacities. Accordingly, 2-D LC was utilized to separate this sample. The first dimension allowed peptides to be separated by their isoelectric point, while the second offered separation on the basis of hydrophobicity. Twelve steps of increasing salt concentrations (0 to 200 mM) were employed. After each salt step, peptides trapped at the tip of the C18 region of the column were subjected to a one-hour gradient. Moreover, the high abundance of serum albumin which usually masks low abundant proteins dictated the need for albumin depletion prior to LC/MALDI/TOFTOF MS analysis. Therefore, blood serum samples were albumin depleted prior to tryptic digestion. Albumin depletion and 2-dimensional LC separation combined with MALDI MS and tandem MS allowed the identification of more than 500 proteins many of which are known to be low abundant proteins. Also, the posttranslational modifications of some of these proteins were assigned by employing database searching of the tandem MS data against certain modifications listed in MASCOT. This study demonstrates several advantages of LC/MALDI/TOFTOF MS including minimize detector saturation, several analysis of same sample spots, decoupling of data from chromatographic separation, and improved quality of MSMS data.

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