Adoption of targeted mass spectrometry (MS) approaches such as multiple reaction monitoring (MRM) to study biological and biomedical questions is well underway in the proteomics community. Successful application depends on the ability to generate reliable assays that uniquely and confidently identify target peptides in a sample. Unfortunately, there is a wide range of criteria being applied to say that an assay has been successfully developed. There is no consensus on what criteria are acceptable and little understanding of the impact of variable criteria on the quality of the results generated. Publications describing targeted MS assays for peptides frequently do not contain sufficient information for readers to establish confidence that the tests work as intended or to be able to apply the tests described in their own labs. Guidance must be developed so that targeted MS assays with established performance can be made widely distributed and applied by many labs worldwide. To begin to address the problems and their solutions, a workshop was held at the National Institutes of Health with representatives from the multiple communities developing and employing targeted MS assays. Participants discussed the analytical goals of their experiments and the experimental evidence needed to establish that the assays they develop work as intended and are achieving the required levels of performance. Using this “fit-for-purpose” approach, the group defined three tiers of assays distinguished by their performance and extent of analytical characterization. Computational and statistical tools useful for the analysis of targeted MS results were described. Participants also detailed the information that authors need to provide in their manuscripts to enable reviewers and readers to clearly understand what procedures were performed and to evaluate the reliability of the peptide or protein quantification measurements reported. This paper presents a summary of the meeting and recommendations.
Various pathogenic clostridia produce binary protein toxins associated with enteric diseases of humans and animals. Separate binding/translocation (B) components bind to a protein receptor on the cell surface, assemble with enzymatic (A) component(s), and mediate endocytosis of the toxin complex. Ultimately there is translocation of A component(s) from acidified endosomes into the cytosol, leading to destruction of the actin cytoskeleton. Our results revealed that CD44, a multifunctional surface protein of mammalian cells, facilitates intoxication by the iota family of clostridial binary toxins. Specific antibody against CD44 inhibited cytotoxicity of the prototypical Clostridium perfringens iota toxin. Versus CD44+ melanoma cells, those lacking CD44 bound less toxin and were dose-dependently resistant to C. perfringens iota, as well as Clostridium difficile and Clostridium spiroforme iota-like, toxins. Purified CD44 specifically interacted in vitro with iota and iota-like, but not related Clostridium botulinum C2, toxins. Furthermore, CD44 knockout mice were resistant to iota toxin lethality. Collective data reveal an important role for CD44 during intoxication by a family of clostridial binary toxins.
Differential 18O/16O stable isotope labeling of peptides that relies on enzyme-catalyzed oxygen exchange at their carboxyl termini in the presence of H218O has been widely used for relative quantitation of peptides/proteins. The role of tryptic proteolysis in bottom-up shotgun proteomics and low reagent costs, has made trypsin-catalyzed 18O post-digestion exchange a convenient and affordable stable isotope labeling approach. However, it is known that trypsin-catalyzed 18O exchange at the carboxyl terminus is in many instances inhomogeneous/incomplete. The extent of the 18O exchange/incorporation fluctuates from peptide to peptide mostly due to variable enzyme-substrate affinity. Thus, accurate calculation and interpretation of peptide ratios are analytically complicated and in some regard deficient. Therefore, a computational approach capable of improved measurement of actual 18O incorporation for each differentially labeled peptide pair is needed. In this regard, we have developed an algorithmic method that relies on the trapezoidal rule to integrate peak intensities of all detected isotopic species across a particular peptide ion over the retention time, which fits the isotopic manifold to Poisson distributions. Optimal values for manifold fitting were calculated and then 18O/16O ratios derived via evolutionary programming. The algorithm is tested using trypsin–catalyzed 18O post-digestion exchange to differentially label bovine serum albumin (BSA) at a priori determined ratios. Both, accuracy and precision are improved utilizing this rigorous mathematical approach. Utilizing this algorithmic technique, we demonstrate the effectiveness of this method to accurately calculate 18O/16O ratios for differentially labeled BSA peptides, by accounting for artifacts caused by a variable degree of post-digestion 18O exchange. We further demonstrate the effectiveness of this method to accurately calculate 18O/16O ratios in a large scale proteomic quantitation of detergent resistant membrane microdomains (DRMMs) isolated from cells expressing wild-type HIV-1 Gag and its non myristylated mutant.
quantitation; 18O/16O stable isotope labeling; variable/incomplete 18O exchange
Effective quantitative profiling of detergent-insoluble membrane proteins using high-throughput mass spectrometry (MS)-based proteomics would allow a better understanding of physiological and pathological processes that take place at the cell surface. To increase the coverage of proteins present in detergent-resistant membrane microdomains (DRMMs), a combination of 16O/18O and isotope coded affinity tags (ICAT) labeling was used in a comparative analysis of detergent-insoluble membrane proteins isolated from rat basophilic leukemia cells (RBL-2H3), with either Triton X-100 or Brij-96. The analysis resulted in the quantification of 738 unique proteins from Triton X-100 and Brij-96 isolated DRMMs, significantly exceeding the number of proteins quantified from either single labeling technique. Twenty-five non-cysteine-containing proteins were quantified, as well as 32 cysteine-containing proteins that would have been missed if either 16O/18O or ICAT labeling had been used exclusively, which illustrate better proteome coverage and enhanced ability to quantitate. The comparative analysis revealed that proteins were more readily extracted using Triton X-100 than Brij-96; however, Triton X-100 also extracted larger quantities of non-DRMMs-associated proteins. This result confirms previous, targeted studies suggesting that DRMMs isolated using Triton X-100 and Brij-96 differ in their protein content.
Quantitative proteomics; combined 16O/18O and ICAT stable isotopic labeling; Triton X-100 and Brij-96 detergent-insoluble membrane proteins
Described is a method that relies on subtractive tissue-directed shot-gun proteomics to identify tumor proteins in the blood of a patient newly diagnosed with cancer. To avoid analytical and statistical biases caused by physiologic variability of protein expression in the human population, this method was applied on clinical specimens obtained from a single patient diagnosed with non-metastatic renal cell carcinoma (RCC). The proteomes extracted from tumor, normal adjacent tissue and pre-operative plasma were analyzed using 2D-LC-MS. The lists of identified proteins were filtered to discover proteins that i) were found in tumor but not normal tissue, ii) were identified in matching plasma, and iii) whose spectral count was higher in tumor tissue than plasma. These filtering criteria resulted in identification of eight tumor proteins in the blood. Subsequent Western-blot analysis confirmed the presence of cadherin-5, cadherin-11, DEAD-box protein-23, and pyruvate kinase) in the blood of the patient under the study, as well as in the blood of four other patients diagnosed with RCC. These results demonstrate the utility of a combined blood/tissue analysis strategy that permits the detection of tumor proteins in the blood of a patient diagnosed with RCC.
Proteomic profiling of membrane proteins is of vital importance in the search for disease biomarkers and drug development. However, the slow pace in this field has resulted mainly from the difficulty to analyze membrane proteins by mass spectrometry (MS). The objective of this investigation was to explore and optimize solubilization of membrane proteins for shotgun membrane proteomics of the CD14 human monocytes by examining different systems that rely on: i) an organic solvent (methanol) ii) an acid-labile detergent 3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate) (PPS), iii) a combination of both agents (methanol + PPS). Solubilization efficiency of different buffers was first compared using bacteriorhodopsin as a model membrane protein. Selected approaches were then applied on a membrane subproteome isolated from a highly enriched human monocyte population that was ~98% positive for CD14 expression by FACS analysis. A methanol-based buffer yielded 194 proteins of which 93 (48%) were mapped as integral membrane proteins. The combination of methanol and acid-cleavable detergent gave similar results; 203 identified proteins of which 93 (46 %) were mapped integral membrane proteins. However, employing PPS a total of 216 proteins of which 75 (35 %) were mapped integral membrane proteins. These results indicate that methanol unaided or in combination with PPS yielded significantly higher membrane protein identification/enrichment than the PPS alone.
CD14 monocyte; Membrane proteins; Solubilization; Methanol; Detergents; LC-MS/MS
Sphingomyelin (SM) and ceramide-phosphoethanolamines (cer-PE) are related lipids present in mammals and insects, respectively. Owing to the critical roles that cer-PE play in eukaryotic cellular function, there is a need to develop methods that provide accurate quantitation of these compounds. Results obtained in this study demonstrate that Drosophila contains cer-PE’s with unsaturated sphingoid base cores as well as low levels of cer-PE’s that possess saturated sphingoid base cores. Specifically, the method developed in this study enabled the quantitation of picogram amounts of cer-PE containing both unsaturated d14:1Δ4 and d16:1Δ4 and saturated d14:0 sphingoid base cores. Using this method cer-PE compounds with both saturated and unsaturated sphingoid base core were initially identified by neutral loss scanning, followed by quantitation using single reaction monitor scans (SRM). The SRM scans measured a product ion originating from the sphingoid base backbone, rather than from the head group, increasing the specificity and the sensitivity of the quantation measurement.
Drosophila; Liquid chromatography; Neutral loss-single ion monitoring scans; Selected reaction monitoring; Quantitation; Ceramide-phosphorylethanolamines; Sphingoid bases
Sphingoid bases, such as unsaturated sphingosine (So) and its corresponding dihydro-saturated species sphinganine (Sa), are present in cell samples in low abundance. This fact combined with their low-to-moderate electrospray ionization (ESI) potential, compared to other sphingolipids such as sphingomyelins, limits their detection and quantitation by liquid chromatography tandem mass spectrometry (LC-MS2). To enhance the ESI efficiency of sphingoid bases, a novel procedure to generate stably derivatized analytes that enhance the LC-MS2 detection of sphingoid bases when analyzed using LC-MS2 was developed. In this method, a ruthenium complex, [4-(N-succimidyloxycarbonyl propyl)-4/-methy-2,2/-bipyridine] bis (2,2/-bipyridine) Ru(11) dihexafluorophosphate, is added directly to a cell extract. This complex reacts with and covalently binds to an amino group within the sphingoid bases. The dicationic nature of the ruthenium ion, enhances the compound’s ionization efficiency resulting in increased LC-MS2 signals for the derivatized sphingoid bases. Consequently, the detection and quantitation of sphingoid bases is greatly improved.
Translocator protein (18-kDa, TSPO1), previously known as the peripheral-type benzodiazepine receptor, is an outer mitochondrial membrane (OMM) protein necessary for cholesterol import and steroid production. We reconstituted the mitochondrial targeting and insertion of TSPO into the OMM to analyze the signals and mechanisms required for this process. Initial studies indicated a formation of a mitochondrial 66-kDa complex through Blue Native-PAGE analysis. The formation of this complex was found to be dependent on the presence of ATP and the cytosolic chaperone Hsp90. Through mutational analysis we identified two areas necessary for TSPO targeting, import, and function: amino acids 103−108 (Schellman motif), which provide the necessary structural orientation for import, and the cholesterol-binding C-terminus required for insertion. Although the Translocase of the Outer Mitochondria Membrane (TOM) complex proteins Tom22 and Tom40 were present in the OMM, the TOM complex did not interact with TSPO. In search of proteins involved in TSPO import, complexes known to interact with TSPO were analyzed by mass spectrometry. The 66-kDa complex formation was found to be dependent on an identified protein, Metaxin 1, for formation and TSPO import. TSPO import into steroidogenic cell mitochondria was increased following treatment of the cells with cAMP. These findings suggest that the initial targeting of TSPO to mitochondria is dependent upon the presence of cytosolic chaperones interacting with the import receptor Tom70. The C-terminus plays an important role in targeting TSPO to mitochondria whereas its import into the OMM is dependent upon the presence of the Schellman motif. Final integration of TSPO into the OMM occurs via its interaction with Metaxin 1. TSPO import into steroidogenic cell mitochondria is regulated by cAMP.
Translocator protein; mitochondria; cholesterol transport; TOM complex; Hsp90 chaperone; cAMP
The purpose of this study was to examine solid tumor heterogeneity on a cellular basis using tissue proteomics that relies on a functional relationship between Laser Capture Microdissection (LCM) and biological mass spectrometry (MS). With the use of LCM, homogeneous regions of cells exhibiting uniform histology were isolated and captured from fresh frozen tissue specimens, which were obtained from a human lymph node containing breast carcinoma metastasis. Six specimens ∼50 000 cell each (three from tumor proper and three from tumor stroma) were collected by LCM. Specimens were processed directly on LCM caps, using sonication in buffered methanol to lyse captured cells, solubilize, and digest extracted proteins. Prepared samples were analyzed by LC/MS/MS resulting in more than 500 unique protein identifications. Decoy database searching revealed a false-positive rate between 5 and 10%. Subcellular localization analysis for stromal cells revealed plasma membrane 14%, cytoplasm 39%, nucleus 11%, extracellular space 27%, and unknown 9%; and tumor cell results were 5%, 58%, 26%, 4%, and 7%, respectively. Western blot analysis confirmed specific linkage of validated proteins to underlying pathology and their potential role in solid tumor heterogeneity. With continued research and optimization of this method including analysis of additional clinical specimens, this approach may lead to an improved understanding of tumor heterogeneity, and serve as a platform for solid tumor biomarker discovery.
laser capture microdissection (LCM); mass spectrometry (MS); solid tumor heterogeneity
A variety of stable isotope labeling techniques have been developed and used in mass spectrometry (MS)-based proteomics, primarily for relative quantitation of changes in protein abundances between two compared samples, but also for qualitative characterization of differentially labeled proteomes. Differential 16O/18O coding relies on the 18O exchange that takes place at the C-terminal carboxyl group of proteolytic fragments, where two 16O atoms are typically replaced by two 18O atoms by enzyme-catalyzed oxygen-exchange in the presence of H218O. The resulting mass shift between differentially labeled peptide ions permits identification, characterization and quantitation of proteins from which the peptides are proteolytically generated. This review focuses on the utility of 16O/18O labeling within the context of mass spectrometry-based proteome research. Different strategies employing 16O/18O are examined in the context of global comparative proteome profiling, targeted subcellular proteomics, analysis of post-translational modifications and biomarker discovery. Also discussed are analytical issues related to this technique, including variable 18O exchange along with advantages and disadvantages of 16O/18O labeling in comparison with other isotope-coding techniques.
18O labeling; enzyme-mediated isotope incorporation; stable isotope labeling; MS-based proteomics; relative protein quantitation; LC/MS/MS
The rapid rise and application of proteomic technologies has resulted in an exponential increase in the number of proteins that have been discovered and presented as ‘potential’ biomarkers for specific diseases. Unfortunately, the number of biomarkers approved for use by the Food and Drug Administration has not risen in likewise manner. While there are a number of reasons for this discrepancy, this glut of ‘potential’ biomarkers also indicates the need for validation methods to confirm or refute their utility in clinical diagnostics. For this reason, the emphasis on developing methods to target and measure the absolute quantity of specific proteins and peptides in complex proteomic samples has grown.
mass spectrometry; biomarker validation; targeted proteomics; multiple-reaction monitoring; AQUA; SISCAPA
A proteomics-based method using stable isotope labeling to assess the relative abundance of peptides or proteins is described. Bradykinin and carbonic anhydrase were labeled with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate, a membrane impermeant reagent that is reactive with primary amines. Specificity of the label to primary amines was demonstrated using tandem mass spectrometry. Also, relative quantitation was achieved by secondary labeling with natural isotopic abundance and stable isotope-labeled methyl iodide. We believe this to be an effective stable isotope-labeling method for quantitative proteomics.
stable isotope labeling; Sulfo-NHS-SS-biotin; proteomics