Accurate quantification of protein expression in biological systems is an increasingly important part of proteomics research. Incorporation of differential stable isotopes in samples for relative protein quantification has been widely used. Stable isotope incorporation at the peptide level using dimethyl labeling is a reliable, cost-effective and undemanding procedure that can be easily automated and applied in high-throughput proteomics experiments. Although alternative multiplex quantitative proteomics approaches introduce isotope labels at the organism level ('stable isotope labeling by amino acids in cell culture' (SILAC)) or enable the simultaneous analysis of eight samples (isobaric tagging for relative and absolute quantification (iTRAQ)), stable isotope dimethyl labeling is advantageous in that it uses inexpensive reagents and is applicable to virtually any sample. We describe in-solution, online and on-column protocols for stable isotope dimethyl labeling of sample amounts ranging from sub-micrograms to milligrams. The labeling steps take approximately 60-90 min, whereas the full protocol including digestion and (two-dimensional) liquid chromatography-mass spectrometry takes approximately 1.5-3 days to complete.

Degradation of cytosolic β-catenin by the APC/Axin1 destruction complex represents the key regulated step of the Wnt pathway. It is incompletely understood how the Axin1 complex exerts its Wnt-regulated function. Here, we examine the mechanism of Wnt signaling under endogenous levels of the Axin1 complex. Our results demonstrate that β-catenin is not only phosphorylated inside the Axin1 complex, but also ubiquinated and degraded via the proteasome, all within an intact Axin1 complex. In disagreement with current views, we find neither a disassembly of the complex nor an inhibition of phosphorylation of Axin1-bound β-catenin upon Wnt signaling. Similar observations are made in primary intestinal epithelium and in colorectal cancer cell lines carrying activating Wnt pathway mutations. Wnt signaling suppresses β-catenin ubiquitination normally occurring within the complex, leading to complex saturation by accumulated phospho-β-catenin. Subsequently, newly synthesized β-catenin can accumulate in a free cytosolic form and engage nuclear TCF transcription factors.

Reversible phosphorylation is a key event in many biological processes and is therefore a much studied phenomenon. The mass spectrometric (MS) analysis of phosphorylation is challenged by the substoichiometric levels of phosphorylation and the lability of the phosphate group in collision-induced dissociation (CID). Here, we review the fragmentation behaviour of phosphorylated peptides in MS and discuss several MS approaches that have been developed to improve and facilitate the analysis of phosphorylated peptides. CID of phosphopeptides typically results in spectra dominated by a neutral loss of the phosphate group. Several proposed mechanisms for this neutral loss and several factors affecting the extent at which this occurs are discussed. Approaches are described to interpret such neutral loss-dominated spectra to identify the phosphopeptide and localize the phosphorylation site. Methods using additional activation, such as MS(3) and multistage activation (MSA), have been designed to generate more sequence-informative fragments from the ion produced by the neutral loss. The characteristics and benefits of these methods are reviewed together with approaches using phosphopeptide derivatization or specific MS scan modes. Additionally, electron-driven dissociation methods by electron capture dissociation (ECD) or electron transfer dissociation (ETD) and their application in phosphopeptide analysis are evaluated. Finally, these techniques are put into perspective for their use in large-scale phosphoproteomics studies.

Temperature-induced cell death is thought to be due to protein denaturation, but the determinants of thermal sensitivity of proteomes remain largely uncharacterized. We developed a structural proteomic strategy to measure protein thermostability on a proteome-wide scale and with domain-level resolution. We applied it to ,, , and human cells, yielding thermostability data for more than 8000 proteins. Our results (i) indicate that temperature-induced cellular collapse is due to the loss of a subset of proteins with key functions, (ii) shed light on the evolutionary conservation of protein and domain stability, and (iii) suggest that natively disordered proteins in a cell are less prevalent than predicted and (iv) that highly expressed proteins are stable because they are designed to tolerate translational errors that would lead to the accumulation of toxic misfolded species.

Changes in protein conformation can affect protein function, but methods to probe these structural changes on a global scale in cells have been lacking. To enable large-scale analyses of protein conformational changes directly in their biological matrices, we present a method that couples limited proteolysis with a targeted proteomics workflow. Using our method, we assessed the structural features of more than 1,000 yeast proteins simultaneously and detected altered conformations for ~300 proteins upon a change of nutrients. We find that some branches of carbon metabolism are transcriptionally regulated whereas others are regulated by enzyme conformational changes. We detect structural changes in aggregation-prone proteins and show the functional relevance of one of these proteins to the metabolic switch. This approach enables probing of both subtle and pronounced structural changes of proteins on a large scale.

Stable isotope labeling is at present one of the most powerful methods in quantitative proteomics. Stable isotope labeling has been performed at both the protein as well as the peptide level using either metabolic or chemical labeling. Here, we present a straightforward and cost-effective triplex quantification method that is based on stable isotope dimethyl labeling at the peptide level. Herein, all proteolytic peptides are chemically labeled at their alpha- and epsilon-amino groups. We use three different isotopomers of formaldehyde to enable the parallel analysis of three different samples. These labels provide a minimum of 4 Da mass difference between peaks in the generated peptide triplets. The method was evaluated based on the quantitative analysis of a cell lysate, using a typical "shotgun" proteomics experiment. While peptide complexity was increased by introducing three labels, still more than 1300 proteins could be identified using 60 microg of starting material, whereby more than 600 proteins could be quantified using at least four peptides per protein. The triplex labeling was further utilized to distinguish specific from aspecific cAMP binding proteins in a chemical proteomics experiment using immobilized cAMP. Thereby, differences in abundance ratio of more than two orders of magnitude could be quantified.

Accumulation of abnormally phosphorylated TDP-43 (pTDP-43) is the main pathology in affected neurons of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Morphological diversity and neuroanatomical distribution of pTDP-43 accumulations allowed classification of FTD cases into at least four different subtypes, which correlate with clinical presentations and genetic causes. To understand the molecular basis of this heterogeneity, we developed SarkoSpin, a new method for extremely pure biochemical isolation of pathological TDP-43. Combining SarkoSpin with mass spectrometry, we revealed proteins beyond TDP-43, which become abnormally insoluble in a disease subtype-specific manner. We show that pTDP-43 extracted from disease brain forms large and stable assemblies of distinct densities and morphologies that correlate with disease subtypes. Importantly, biochemically extracted pTDP-43 assemblies displayed differential neurotoxicity and seeding that correlated with disease duration of FTLD patients. Our data indicate that disease heterogeneity may originate from alternate pathological TDP-43 conformations, reminiscent of prion strains. 5 developed SarkoSpin, a novel and simple extraction method for physical separation of pathological TDP-43 from more than 99% of total protein mass including the extreme bulk of physiological, monomeric and oligomeric 11 TDP-43. Using SarkoSpin on brain cortical samples from over 80 patients, we found that TDP-43 forms large and buoyant assemblies of distinct densities, polyubiquitination levels and morphologies that correlate with specific neuropathological classifications. Importantly, coupling SarkoSpin with mass spectrometry, we illustrate that a specific subset of proteins, beyond TDP-43, become insoluble in each disease subtype. These proteins are rarely co-aggregated with pTDP-43 and most likely represent a downstream effect of TDP-43 pathology. One of these proteins depicts a distinct astrocytic reaction discriminating FTLD-TDP-A from FTLD-TDP-C patients, illustrating divergent pathogenic mechanisms within these two disease subtypes. Most importantly, we show evidence that SarkoSpin extracted pTDP-43 assemblies exhibit cytotoxicity and protein seeding ability. Remarkably, pathological aggregates extracted from FTLD-TDP-A were significantly more cyto-and neurotoxic than those extracted from FTLD-TDP-C, thereby correlating with the significant difference in disease duration between these two subtypes. Collectively, our data demonstrate that ALS and FTLD heterogeneity is consistently reflected in the biochemical, neurotoxic and seeding properties of TDP-43 and the associated insoluble proteome. We propose that alternative TDP-43 pathological conformations may underlie the diversity of TDP-43 proteinopathies, reminiscent of prion strains 33,34. Results Summary of patient cohort and characterization of FTLD-TDP-A and FTLD-TDP-C cases Brain cortical samples from over 80 patients, including control patients with no apparent CNS pathology or with non-TDP...

Protein structural changes induced by external perturbations or internal cues can profoundly influence protein activity and thus modulate cellular physiology. A number of biophysical approaches are available to probe protein structural changes, but these are not applicable to a whole proteome in a biological extract. Limited proteolysis-coupled mass spectrometry (LiP-MS) is a recently developed proteomics approach that enables the identification of protein structural changes directly in their complex biological context on a proteome-wide scale. After perturbations of interest, proteome extracts are subjected to a double-protease digestion step with a nonspecific protease applied under native conditions, followed by complete digestion with the sequence-specific protease trypsin under denaturing conditions. This sequential treatment generates structure-specific peptides amenable to bottom-up MS analysis. Next, a proteomics workflow involving shotgun or targeted MS and label-free quantification is applied to measure structure-dependent proteolytic patterns directly in the proteome extract. Possible applications of LiP-MS include discovery of perturbation-induced protein structural alterations, identification of drug targets, detection of disease-associated protein structural states, and analysis of protein aggregates directly in biological samples. The approach also enables identification of the specific protein regions involved in the structural transition or affected by the binding event. Sample preparation takes approximately 2 d, followed by one to several days of MS and data analysis time, depending on the number of samples analyzed. Scientists with basic biochemistry training can implement the sample preparation steps. MS measurement and data analysis require a background in proteomics.