Proteins from silver-stained gels can be digested enzymatically and the resulting peptide analyzed and sequenced by mass spectrometry. Standard proteins yield the same peptide maps when extracted from Coomassie- and silver-stained gels, as judged by electrospray and MALDI mass spectrometry. The low nanogram range can be reached by the protocols described here, and the method is robust. A silver-stained one-dimensional gel of a fraction from yeast proteins was analyzed by nano-electrospray tandem mass spectrometry. In the sequencing, more than 1000 amino acids were covered, resulting in no evidence of chemical modifications due to the silver staining procedure. Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining. This work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.

In-gel digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometry (MS)-driven proteomics. The 10-year-old recipe by Shevchenko et al. has been optimized to increase the speed and sensitivity of analysis. The protocol is for the in-gel digestion of both silver and Coomassie-stained protein spots or bands and can be followed by MALDI-MS or LC-MS/MS analysis to identify proteins at sensitivities better than a few femtomoles of protein starting material.

To identify CAP3 and CAP4, components of the CD95 (Fas/APO-1) death-inducing signaling complex, we utilized nano-electrospray tandem mass spectrometry, a recently developed technique to sequence femtomole quantities of polyacrylamide gel-separated proteins. Interestingly, CAP4 encodes a novel 55 kDa protein, designated FLICE, which has homology to both FADD and the ICE/CED-3 family of cysteine proteases. FLICE binds to the death effector domain of FADD and upon overexpression induces apoptosis that is blocked by the ICE family inhibitors, CrmA and z-VAD-fmk. CAP3 was identified as the FLICE prodomain which likely remains bound to the receptor after proteolytic activation. Taken together, this is unique biochemical evidence to link a death receptor physically to the proapoptotic proteases of the ICE/CED-3 family.

Activation of the transcription factor nuclear factor kappa B (NF-kappaB) is controlled by sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit IkappaB. A large multiprotein complex, the IkappaB kinase (IKK) signalsome, was purified from HeLa cells and found to contain a cytokine-inducible IkappaB kinase activity that phosphorylates IkappaB-alpha and IkappaB-beta. Two components of the IKK signalsome, IKK-1 and IKK-2, were identified as closely related protein serine kinases containing leucine zipper and helix-loop-helix protein interaction motifs. Mutant versions of IKK-2 had pronounced effects on RelA nuclear translocation and NF-kappaB-dependent reporter activity, consistent with a critical role for the IKK kinases in the NF-kappaB signaling pathway.

Accurate profiling of lipidomes relies upon the quantitative and unbiased recovery of lipid species from analyzed cells, fluids, or tissues and is usually achieved by two-phase extraction with chloroform. We demonstrated that methyl-tert-butyl ether (MTBE) extraction allows faster and cleaner lipid recovery and is well suited for automated shotgun profiling. Because of MTBE's low density, lipid-containing organic phase forms the upper layer during phase separation, which simplifies its collection and minimizes dripping losses. Nonextractable matrix forms a dense pellet at the bottom of the extraction tube and is easily removed by centrifugation. Rigorous testing demonstrated that the MTBE protocol delivers similar or better recoveries of species of most all major lipid classes compared with the "gold-standard" Folch or Bligh and Dyer recipes. Recent developments in mass spectrometric technology enabled the comprehensive characterization of eukaryotic lipidomes, fostering the molecular biology of lipids and metabolism-related disorders (reviewed in Refs. 1-4). Typically, lipidome profiling by mass spectrometry proceeds along LC-MS or shotgun approaches. The former identifies and quantifies lipid species preseparated by normal or reversed-phase chromatography coupled online to a mass spectrometer, which is capable of fast acquisition of MS or MS/MS spectra (5-8). In contrast, in shotgun lipidomics, total lipid extracts are infused directly into a mass spectrometer, and the molecular characterization of lipid species relies either on the accurately determined m/z of precursor ions (9) or on the detection of specific fragment ions or neutral losses in tandem mass spectrometric experiments (1, 9-12).Regardless of the analytical approach used, its success depends on the completeness of the extraction of lipids from corresponding cells, fluids, or tissues. Lipids of all major classes could be recovered via chloroform/methanol extraction, typically according to the Folch, Lees, and Sloane Stanley (13) or Bligh and Dyer (14) recipes (15), in which they are mostly enriched in the chloroform phase.Electrospray mass spectrometry, a major tool for analyzing complex lipidomes, is particularly sensitive towards the quality of lipid extracts. Coextracted components of biological matrix and salts (often, without further definition, termed background) affect both the sensitivity and specificity of lipid analysis. Often, abundant background ions obscure lipid precursors, and their MS/MS spectra are densely populated with "ghost" peaks and abundant chemical noise. Adducts with common background cations (sodium, potassium) and anions (chloride) increase the ambiguity of molecular species assignment and affect the accuracy of quantitative determination.Because of the higher density of chloroform compared with a water/methanol mixture, it forms the lower phase of the two-phase partitioning system. While collecting the chloroform fraction, a glass pipette or a needle of the pipetting robot reaches it through a voluminous layer...

Molecular analysis of complex biological structures and processes increasingly requires sensitive methods for protein sequencing. Electrospray mass spectrometry has been applied to the high-sensitivity sequencing of short peptides, but technical difficulties have prevented similar success with gel-isolated proteins. Here we report a simple and robust technique for the sequencing of proteins isolated by polyacrylamide gel electrophoresis, using nano-electrospray tandem mass spectrometry. As little as 5 ng protein starting material on Coomassie- or silver-stained gels can be sequenced. Multiple-sequence stretches of up to 16 amino acids are obtained, which identify the protein unambiguously if already present in databases or provide information to clone the corresponding gene. We have applied this method to the sequencing and cloning of a protein which inhibits the proliferation of capillary endothelial cells in vitro and thus may have potential antiangiogenic effects on solid tumours.

Although the transcriptome, proteome, and interactome of several eukaryotic model organisms have been described in detail, lipidomes remain relatively uncharacterized. Using Saccharomyces cerevisiae as an example, we demonstrate that automated shotgun lipidomics analysis enabled lipidome-wide absolute quantification of individual molecular lipid species by streamlined processing of a single sample of only 2 million yeast cells. By comparative lipidomics, we achieved the absolute quantification of 250 molecular lipid species covering 21 major lipid classes. This analysis provided Ϸ95% coverage of the yeast lipidome achieved with 125-fold improvement in sensitivity compared with previous approaches. Comparative lipidomics demonstrated that growth temperature and defects in lipid biosynthesis induce ripple effects throughout the molecular composition of the yeast lipidome. This work serves as a resource for molecular characterization of eukaryotic lipidomes, and establishes shotgun lipidomics as a powerful platform for complementing biochemical studies and other systems-level approaches.fatty acid elongation ͉ S. cerevisiae ͉ shotgun lipidomics T he lipidome of eukaryotic cells consists of hundreds to thousands of individual lipid species that constitute membranes, store metabolic energy and function as bioactive molecules (1-3). Despite the extensive characterization of proteins, their association into complexes and activities (4-6), it is still difficult to assess how perturbations within the lipid metabolic network affect the full lipidome of cells. This work shows that lipidome-wide quantification of individual molecular lipid species (molecules with defined chemical structure) by absolute quantification (expressed in mol or mol%) provides a new approach to relate lipidomics and functional genomics studies.The yeast Saccharomyces cerevisiae serves as a prime model organism for studying the molecular organization and regulatory circuitry of eukaryotic lipidomes (7-9). It uses a relatively simple and conserved network of lipid metabolic pathways (Fig. 1) that synthesize a few hundred molecular lipid species constituting its full lipidome (3). The lipidome diversity is primarily determined by the fatty acid synthase (10), the ⌬-9 desaturase (11) and the fatty acid elongation complex (12) that produce only saturated or mono-unsaturated fatty acids having 10 to 26 carbon atoms for the biosynthesis of glycerolipids, glycerophospholipids, and sphingolipids. Importantly, several metabolic conversions interlink sphingolipid, glycerophospholipid, and glycerolipid metabolism such that any perturbation within the metabolic network is prone to induce lipidome-wide ripple effects. Remarkably, numerous genes involved in lipid metabolism and trafficking can be mutated or deleted without apparent physiological consequences (Fig. 1).Despite remarkable methodological advances, lipidomics seldom complements functional genomics efforts owing to three major factors. First, analysis of glycerophospholipids and sphingolipids requires ...

Telomerase is a ribonucleoprotein enzyme essential for the replication of chromosome termini in most eukaryotes. Telomerase RNA components have been identified from many organisms, but no protein component has been demonstrated to catalyze telomeric DNA extension. Telomerase was purified from Euplotes aediculatus, a ciliated protozoan, and one of its proteins was partially sequenced by nanoelectrospray tandem mass spectrometry. Cloning and sequence analysis of the corresponding gene revealed that this 123-kilodalton protein (p123) contains reverse transcriptase motifs. A yeast (Saccharomyces cerevisiae) homolog was found and subsequently identified as EST2 (ever shorter telomeres), deletion of which had independently been shown to produce telomere defects. Introduction of single amino acid substitutions within the reverse transcriptase motifs of Est2 protein led to telomere shortening and senescence in yeast, indicating that these motifs are important for catalysis of telomere elongation in vivo. In vitro telomeric DNA extension occurred with extracts from wild-type yeast but not from est2 mutants or mutants deficient in telomerase RNA. Thus, the reverse transcriptase protein fold, previously known to be involved in retroviral replication and retrotransposition, is essential for normal chromosome telomere replication in diverse eukaryotes.