LC-MALDI MS and MS/MS--an efficient tool in proteome analysis.

Liquid chromatography-matrix-assisted laser desorption/ionization mass spectrometry represents a sensitive, hyphenated MS- and MS/MS-technique with a broad range of applications in all areas ofproteome analysis. Whereas a number of interface types have been developed for coupling MALDI MS and liquid chromatography, in this chapter selected on-line and off-line types and techniques will be discussed with respect to their individual properties and performance. The technique is especially attractive in off-line mode where LC-separation and MS analyses are decoupled and each step can be performed at its individual optimum. Different speed of chromatographic separation and achievement of S/N criteria in MS or MS/MS mode can be optimized independently by individual adjustment of specific operating parameters. This flexibility makes LC-MALDI MS attractive for the analysis of peptide mixtures from low to medium complexity. Using sequential MS analysis of parallel LC runs (multiplexing), even highly complex samples can be handled. Quantitation at the MS and MS/MS level can be accomplished by a variety of labeling techniques, where the predominant formation of singly charged ions in MALDI alleviates the assignment of isotopomers. After discussing the level of complementarity between LC-MALDI and LC-ESI MS, selected applications of LC-MALDI MS are presented. Examples of membrane protein analysis applying 1D SDS PAGE are discussed in detail as well as applications in protein interaction analysis. These application examples clearly show that in all respects LC-MALDI MS and MS/MS are flexible and sensitive techniques which can be adapted to a wide range of different workflows.

Analysis of dynamic changes in post-translational modifications of human histones during cell cycle by mass spectrometry.

The N-terminal tails of the four core histones are subject to several types of covalent post-translational modifications that have specific roles in regulating chromatin structure and function. Here we present an extensive analysis of the core histone modifications occurring through the cell cycle. Our MS experiments characterized the modification patterns of histones from HeLa cells arrested in phase G1, S, and G2/M. For all core histones, the modifications in the G1 and S phases were largely identical but drastically different during mitosis. Modification changes between S and G2/M phases were quantified using the SILAC (stable isotope labeling by amino acids in cell culture) approach. Most striking was the mitotic phosphorylation on histone H3 and H4, whereas phosphorylation on H2A was constant during the cell cycle. A loss of acetylation was observed on all histones in G2/M-arrested cells. The pattern of cycle-dependent methylation was more complex: during G2/M, H3 Lys27 and Lys36 were decreased, whereas H4 Lys20 was increased. Our results show that mitosis was the period of the cell cycle during which many modifications exhibit dynamic changes.

Proteomics-based target identification: bengamides as a new class of methionine aminopeptidase inhibitors.

LAF389 is a synthetic analogue of bengamides, a class of marine natural products that produce inhibitory effects on tumor growth in vitro and in vivo. A proteomics-based approach has been used to identify signaling pathways affected by bengamides. LAF389 treatment of cells resulted in altered mobility of a subset of proteins on two-dimensional gel electrophoresis. Detailed analysis of one of the proteins, 14-3-3gamma, showed that bengamide treatment resulted in retention of the amino-terminal methionine, suggesting that bengamides directly or indirectly inhibited methionine aminopeptidases (MetAps). Both known MetAps are inhibited by LAF389. Short interfering RNA suppression of MetAp2 also altered amino-terminal processing of 14-3-3gamma. A high resolution structure of human MetAp2 co-crystallized with a bengamide shows that the compound binds in a manner that mimics peptide substrates. Additionally, the structure reveals that three key hydroxyl groups on the inhibitor coordinate the di-cobalt center in the enzyme active site.

Intact protein analysis by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry.

Direct tandem mass spectrometric (MS/MS) analysis of small, singly charged protein ions by tandem time-of-flight mass spectrometry (TOFMS) is demonstrated for proteins up to a molecular mass of 12 kDa. The MALDI-generated singly charged precursor ions predominantly yield product ions resulting from metastable fragmentation at aspartyl and prolyl residues. Additional series of C-terminal sequence ions provide in some cases sufficient information for protein identification. The amount of sample required to obtain good quality spectra is in the high femtomolar to low picomolar range. Within this range, MALDI-MS/MS using TOF/TOF trade mark ion optics now provides the opportunity for direct protein identification and partial characterization without prior enzymatic hydrolysis.