Jacek R. Wiśniewski

Combination of FASP and StageTip-Based Fractionation Allows In-Depth Analysis of the Hippocampal Membrane Proteome

Membrane proteomics is challenging because the desirable strong detergents are incompatible with downstream analysis. Recently, we demonstrated efficient removal of SDS by the filter aided sample preparation method (FASP). Here we combine FASP with our previously described small-scale membrane enrichment protocol. Analysis of a single mouse hippocampus enables identification of more than 1000 membrane proteins in a single LC-MS/MS run without protein or peptide prefractionation. To extend proteome coverage, we developed a simple anion exchange fractionation method in a StageTip format. When separating peptides into six fractions, a duplicate analysis resulted in identification of 4206 proteins of which 64% were membrane proteins. This data set covers 83% of glutamate and GABA receptor subunits identified in hippocampus in the Allen Brain Atlas and adds further isoforms. The combined method provides a streamlined protocol for rapid and sensitive membrane proteome mapping. We also provide a generic protocol for combining FASP with StageTip-based ion exchange fractionation, which is generally applicable to proteome analysis.

Ultradeep Human Phosphoproteome Reveals a Distinct Regulatory Nature of Tyr and Ser/Thr-Based Signaling

Regulatory protein phosphorylation controls normal and pathophysiological signaling in eukaryotic cells. Despite great advances in mass-spectrometry-based proteomics, the extent, localization, and site-specific stoichiometry of this posttranslational modification (PTM) are unknown. Here, we develop a stringent experimental and computational workflow, capable of mapping more than 50,000 distinct phosphorylated peptides in a single human cancer cell line. We detected more than three-quarters of cellular proteins as phosphoproteins and determined very high stoichiometries in mitosis or growth factor signaling by label-free quantitation. The proportion of phospho-Tyr drastically decreases as coverage of the phosphoproteome increases, whereas Ser/Thr sites saturate only for technical reasons. Tyrosine phosphorylation is maintained at especially low stoichiometric levels in the absence of specific signaling events. Unexpectedly, it is enriched on higher-abundance proteins, and this correlates with the substrate KM values of tyrosine kinases. Our data suggest that P-Tyr should be considered a functionally separate PTM of eukaryotic proteomes.

Consecutive Proteolytic Digestion in an Enzyme Reactor Increases Depth of Proteomic and Phosphoproteomic Analysis

Analytical advantages of using multiple enzymes for sample digestion (MED), primarily an increase of sequence coverage, have been reported in several studies. However, this approach is only rarely used, mainly because it requires additional sample and mass spectrometric measurement time. We have previously described Filter Aided Sample Preparation (FASP), a type of proteomic reactor, in which samples dissolved in sodium dodecyl sulfate (SDS) are digested in an ultrafiltration unit. In FASP, such as in any other preparation protocol, a portion of sample remains after digestion and peptide elution. Making use of this fact, we here develop a protocol enabling consecutive digestion of the sample with two or three enzymes. By use of the FASP method, peptides are liberated after each digestion step and remaining material is subsequently cleaved with the next proteinase. We observed excellent performance of the ultrafiltration devices in this mode, allowing efficient separation of orthogonal populations of peptides, resulting in an increase in the numbers of identified peptides and proteins. At the low microgram level, we found that the consecutive use of endoproteinases LysC and trypsin enabled identification of up to 40% more proteins and phosphorylation sites in comparison to the commonly used one-step tryptic digestion. MED-FASP offers efficient exploration of previously unused sample material, increasing depth of proteomic analyses and sequence coverage.

Extensive quantitative remodeling of the proteome between normal colon tissue and adenocarcinoma

We report a proteomic analysis of microdissected material from formalin‐fixed and paraffin‐embedded colorectal cancer, quantifying >7500 proteins between patient matched normal mucosa, primary carcinoma, and nodal metastases. Expression levels of 1808 proteins changed significantly between normal and cancer tissues, a much larger fraction than that reported in transcript‐based studies. Tumor cells exhibit extensive alterations in the cell‐surface and nuclear proteomes. Functionally similar changes in the proteome were observed comparing rapidly growing and differentiated CaCo‐2 cells. In contrast, there was minimal proteomic remodeling between primary cancer and metastases, suggesting that no drastic proteome changes are necessary for the tumor to propagate in a different tissue context. Additionally, we introduce a new way to determine protein copy numbers per cell without protein standards. Copy numbers estimated in enterocytes and cancer cells are in good agreement with CaCo‐2 and HeLa cells and with the literature data. Our proteomic data set furthermore allows mapping quantitative changes of functional protein classes, enabling novel insights into the biology of colon cancer.

A 'proteomic ruler' for protein copy number and concentration estimation without spike-in standards

Absolute protein quantification using mass spectrometry (MS)-based proteomics delivers protein concentrations or copy numbers per cell. Existing methodologies typically require a combination of isotope-labeled spike-in references, cell counting, and protein concentration measurements. Here we present a novel method that delivers similar quantitative results directly from deep eukaryotic proteome datasets without any additional experimental steps. We show that the MS signal of histones can be used as a “proteomic ruler” because it is proportional to the amount of DNA in the sample, which in turn depends on the number of cells. As a result, our proteomic ruler approach adds an absolute scale to the MS readout and allows estimation of the copy numbers of individual proteins per cell. We compare our protein quantifications with values derived via the use of stable isotope labeling by amino acids in cell culture and protein epitope signature tags in a method that combines spike-in protein fragment standards with precise isotope label quantification. The proteomic ruler approach yields quantitative readouts that are in remarkably good agreement with results from the precision method. We attribute this surprising result to the fact that the proteomic ruler approach omits error-prone steps such as cell counting or protein concentration measurements. The proteomic ruler approach is readily applicable to any deep eukaryotic proteome dataset—even in retrospective analysis—and we demonstrate its usefulness with a series of mouse organ proteomes.

Mapping N-Glycosylation Sites across Seven Evolutionarily Distant Species Reveals a Divergent Substrate Proteome Despite a Common Core Machinery

N-linked glycosylation is an important posttranslational modification in all eukaryotes, but little is known about the N-glycoproteomes in nonmammalian systems. Here, we measure N-glycoproteomes of the major model organisms Arabidopsis thaliana, Schizosaccharomyces pombe, Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and Danio rerio, representatively spanning the eukaryotic domain of life. The number of detected N-glycosylation sites varied between 425 in fission yeast, 516 in budding yeast, 1,794 in worm, 2,186 in plant, 2,229 in fly, and 2,254 in zebrafish. We find that all eukaryotic N-glycoproteomes have invariant characteristics including sequence recognition patterns, structural constraints, and subcellular localization. However, a surprisingly large percentage of the N-glycoproteome evolved after the phylogenetic divergences between plants, fungi, nematodes, insects, and vertebrates. Many N-glycosylated proteins coevolved with the rise of extracellular processes that are specific within corresponding phylogenetic groups and essential for organismal development, body growth, and organ formation.

Proteomic Mapping of Brain Plasma Membrane Proteins

Proteomics is potentially a powerful technology for elucidating brain function and neurodegenerative diseases. So far, the brain proteome has generally been analyzed by two-dimensional gel electrophoresis, which usually leads to the complete absence of membrane proteins. We describe a proteomic approach for profiling of plasma membrane proteins from mouse brain. The procedure consists of a novel method for extraction and fractionation of membranes, on-membrane digestion, diagonal separation of peptides, and high-sensitivity analysis by advanced MS. Breaking with the classical plasma membrane fractionation approach, membranes are isolated without cell compartment isolation, by stepwise depletion of nonmembrane molecules from entire tissue homogenate by high-salt, carbonate, and urea washes followed by treatment of the membranes with sublytic concentrations of digitonin. Plasma membrane is further enriched by of density gradient fractionation and protein digested on-membrane by endoproteinase Lys-C. Released peptides are separated, fractions digested by trypsin, and analyzed by LC-MS/MS. In single experiments, the developed technology enabled identification of 862 proteins from 150 mg of mouse brain cortex. Further development and miniaturization allowed analysis of 15 mg of hippocampus, revealing 1,685 proteins. More that 60% of the identified proteins are membrane proteins, including several classes of ion channels and neurotransmitter receptors. Our work now allows in-depth study of brain membrane proteomes, such as of mouse models of neurological disease.

Proteomic workflow for analysis of archival formalin‐fixed and paraffin‐embedded clinical samples to a depth of 10 000 proteins

Archival formalin‐fixed and paraffin‐embedded clinical samples represent a very diverse source of material for proteomic investigation of diseases, often with follow‐up patient information. Here, we describe an analytical workflow for analysis of laser‐capture microdissected formalin‐fixed and paraffin‐embedded samples that allows studying proteomes to a depth of 10 000 proteins per sample.

HysTag—A Novel Proteomic Quantification Tool Applied to Differential Display Analysis of Membrane Proteins From Distinct Areas of Mouse Brain

A novel isotopically labeled cysteine-tagging and complexity-reducing reagent, called HysTag, has been synthesized and used for quantitative proteomics of proteins from enriched plasma membrane preparations from mouse fore- and hindbrain. The reagent is a 10-mer derivatized peptide, H2N-(His)6-Ala-Arg-Ala-Cys(2-thiopyridyl disulfide)-CO2H, which consists of four functional elements: i) an affinity ligand (His6-tag), ii) a tryptic cleavage site (-Arg-Ala-), iii) Ala-9 residue that contains four (d4) or no (d0) deuterium atoms, and iv) a thiol-reactive group (2-thiopyridyl disulfide). For differential analysis cysteine residues in the compared samples are modified using either (d4) or (d0) reagent. The HysTag peptide is preserved in Lys-C digestion of proteins and allows charge-based selection of cysteine-containing peptides, whereas subsequent tryptic digestion reduces the labeling group to a di-peptide, which does not hinder effective fragmentation. Furthermore, we found that tagged peptides containing Ala-d4 co-elute with their d0-labeled counterparts. To demonstrate effectiveness of the reagent, a differential analysis of mouse forebrain versus hindbrain plasma membranes was performed. Enriched plasma membrane fractions were partially denatured, reduced, and reacted with the reagent. Digestion with endoproteinase Lys-C was carried out on nonsolubilized membranes. The membranes were sedimented by ultra centrifugation, and the tagged peptides were isolated by Ni2+ affinity or cation-exchange chromatography. Finally, the tagged peptides were cleaved with trypsin to release the histidine tag (residues 1–8 of the reagent) followed by liquid chromatography tandem mass spectroscopy for relative protein quantification and identification. A total of 355 unique proteins were identified, among which 281 could be quantified. Among a large majority of proteins with ratios close to one, a few proteins with significant quantitative changes were retrieved. The HysTag offers advantages compared with the isotope-coded affinity tag reagent, because the HysTag reagent is easy to synthesize, economical due to use of deuterium instead of 13C isotope label, and allows robust purification and flexibility through the affinity tag, which can be extended to different peptide functionalities.

Quantification of the N-glycosylated Secretome by Super-SILAC During Breast Cancer Progression and in Human Blood Samples

Cells secrete a large number of proteins to communicate with their surroundings. Furthermore, plasma membrane proteins and intracellular proteins can be released into the extracellular space by regulated or non-regulated processes. Here, we profiled the supernatant of 11 cell lines that are representative of different stages of breast cancer development by specifically capturing N-glycosylated peptides using the N-glyco FASP technology. For accurate quantification we developed a super-SILAC mix from several labeled breast cancer cell lines and used it as an internal standard for all samples. In total, 1398 unique N-glycosylation sites were identified and quantified. Enriching for N-glycosylated peptides focused the analysis on classically secreted and membrane proteins. N-glycosylated secretome profiles correctly clustered the different cell lines to their respective cancer stage, suggesting that biologically relevant differences were detected. Five different profiles of glycoprotein dynamics during cancer development were detected, and they contained several proteins with known roles in breast cancer. We then used the super-SILAC mix in plasma, which led to the quantification of a large number of the previously identified N-glycopeptides in this important body fluid. The combination of quantifying the secretome of cancer cell lines and of human plasma with a super-SILAC approach appears to be a promising new approach for finding markers of disease.