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The most widely used cancer animal model is the human-murine tumor xenograft. Unbiased molecular dissection of tumor parenchyma versus stroma in human-murine xenografts is critical for elucidating dysregulated protein networks/pathways and developing therapeutics that may target these two functionally codependent compartments. Although antibody-reliant technologies (e.g., immunohistochemistry, imaging mass cytometry) are capable of distinguishing tumor-proper versus stromal proteins, the breadth or extent of targets is limited. Here, we report an antibody-free targeted cross-species glycoproteomic (TCSG) approach that enables direct dissection of human tumor parenchyma from murine tumor stroma at the molecular/protein level in tumor xenografts at a selectivity rate presently unattainable by other means. This approach was used to segment/dissect and obtain the protein complement phenotype of the tumor stroma and parenchyma of the metastatic human lung adenocarcinoma A549 xenograft, with no need for tissue microdissection prior to mass-spectrometry analysis. An extensive molecular map of the tumor proper and the associated microenvironment was generated along with the top functional N-glycosylated protein networks enriched in each compartment. Importantly, immunohistochemistry-based cross-validation of selected parenchymal and stromal targets applied on human tissue samples of lung adenocarcinoma and normal adjacent tissue is indicative of a noteworthy translational capacity for this unique approach that may facilitate identifications of novel targets for next generation antibody therapies and development of real time preclinical tumor models.

To ensure accuracy of UGT1A1 (TA)n (rs3064744) genotyping for use in pharmacogenomics-based irinotecan dosing, we tested the concordance of several commonly used genotyping technologies. Heuristic genotype groupings and principal component analysis demonstrated concordance for Illumina sequencing, fragment analysis, and fluorescent PCR. However, Illumina sequencing and fragment analysis returned a range of fragment sizes, likely arising due to PCR &ldquo, slippage&rdquo, Direct sequencing was accurate, but this method led to ambiguous electrophoregrams, hampering interpretation of heterozygotes. Gel sizing, pyrosequencing, and array-based technologies were less concordant. Pharmacoscan genotyping was concordant, but it does not ascertain (TA)8 genotypes that are common in African populations. Method-based genotyping differences were also observed in the publication record (p &lt, 0.0046), although fragment analysis and direct sequencing were concordant (p = 0.11). Genotyping errors can have significant consequences in a clinical setting. At the present time, we recommend that all genotyping for this allele be conducted with fluorescent PCR (fPCR).

There is intense interest in developing therapeutic strategies for RAS proteins, the most frequently mutated oncoprotein family in cancer. Development of effective anti-RAS therapies will be aided by the greater appreciation of RAS isoform-specific differences in signaling events that support neoplastic cell growth. However, critical issues that require resolution to facilitate the success of these efforts remain. In particular, the use of well-validated anti-RAS antibodies is essential for accurate interpretation of experimental data. We evaluated 22 commercially available anti-RAS antibodies with a set of distinct reagents and cell lines for their specificity and selectivity in recognizing the intended RAS isoforms and mutants. Reliability varied substantially. For example, we found that some pan- or isoform-selective anti-RAS antibodies did not adequately recognize their intended target or showed greater selectivity for another; some were valid for detecting G12D and G12V mutant RAS proteins in Western blotting, but none were valid for immunofluorescence or immunohistochemical analyses; and some antibodies recognized nonspecific bands in lysates from "Rasless" cells expressing the oncoprotein BRAFV600E Using our validated antibodies, we identified RAS isoform-specific siRNAs and shRNAs. Our results may help to ensure the accurate interpretation of future RAS studies.

Immunoaffinity enrichment of peptides coupled to targeted, multiple reaction monitoring-mass spectrometry (immuno-MRM) has recently been developed for quantitative analysis of peptide and protein expression. As part of this technology, antibodies are generated to short, linear, tryptic peptides that are well-suited for detection by mass spectrometry. Despite its favorable analytical performance, a major obstacle to widespread adoption of immuno-MRM is a lack of validated affinity reagents because commercial antibody suppliers are reluctant to commit resources to producing anti-peptide antibodies for immuno-MRM while the market is much larger for conventional technologies, especially Western blotting and ELISA. Part of this reluctance has been the concern that affinity reagents generated to short, linear, tryptic peptide sequences may not perform well in traditional assays that detect full-length proteins. In this study, we test the feasibility and success rates of generating immuno-MRM monoclonal antibodies (mAbs) (targeting tryptic peptide antigens) that are also compatible with conventional, protein-based immuno-affinity technologies. We generated 40 novel, peptide immuno-MRM assays and determined that the cross-over success rates for using immuno-MRM monoclonals for Western blotting is 58% and for ELISA is 43%, which compare favorably to cross-over success rates amongst conventional immunoassay technologies. These success rates could most likely be increased if conventional and immuno-MRM antigen design strategies were combined, and we suggest a workflow for such a comprehensive approach. Additionally, the 40 novel immuno-MRM assays underwent fit-for-purpose analytical validation, and all mAbs and assays have been made available as a resource to the community via the Clinical Proteomic Tumor Analysis Consortium's (CPTAC) Antibody (http://antibodies.cancer.gov) and Assay Portals (http://assays.cancer.gov), respectively. This study also represents the first determination of the success rate (92%) for generating mAbs for immuno-MRM using a recombinant B cell cloning approach, which is considerably faster than the traditional hybridoma approach.

We tested the hypothesis that differences in the low-molecular-weight (500-20,000 Da) proteomic profile of plasma may be detectable between members of a protein C-deficient family who have suffered thrombotic events before age 40 compared to family members without a history of venous thrombosis. Unfractionated plasma samples from members of a previously described large thrombophilic kindred with type I protein C deficiency were applied to ProteinChip weak cation exchange interaction arrays (WCX2; Ciphergen Biosystems, Fremont, CA, USA) and subjected to SELDI-TOF (surface-enhanced laser desorption/ionization time-of-flight) mass spectrometry using the Ciphergen PBSII ProteinChip System (Ciphergen Biosystems). Profiles were analyzed by a boosted decision-tree algorithm. When individuals who had presented with deep venous thrombosis (DVT) before the age of 40 (n = 21) were compared to age-matched, healthy family members (n = 50), the proteomic patterns defined by the decision-tree analysis could classify the entity of DVT before age 40 with 67% sensitivity, at a specificity of 86%. When a small group of cases with history of superficial venous thrombosis (n = 6) was added to the case group, the sensitivity was 87.5% at a specificity of 80%. These data support the hypothesis that members of the protein C deficient Vermont kindred II who suffer a thrombotic event before age 40 display significant differences in low-molecular-weight proteomics profile compared to those who remain disease-free. This is the first study to apply SELDI-TOF technology in conjunction with a bioinformatics tool to analyze low-molecular-weight proteomic patterns in patients with venous thrombosis.

Providencia stuartii nosocomial urinary tract infections occurring in the same hospital over an 18-month period of retrospective study were shown, by serotyping and biotyping, to have been caused by two endemic strains. Two episodes, involving 38 patients in one ward and 11 patients in another, were caused by a mannitol-positive strain of serotype O55. Transmission of the strain through the movements of one patient appeared to have been the basis for the introduction of the agent from one ward to the other. In another episode, involving two patients in a third ward, the infections were caused by a mannitol-negative strain of serotype O49. The study demonstrated the usefulness of serotyping and biotyping in epidemiological studies of infections caused by P. stuartii.

Both urease-positive and urease-negative Proteeae isolated from cross-infected patients in the same hospitals and, in three cases, from the same patients were examined for their biochemical reactions and somatic (O-) antigens. All isolates gave the same reactions in 17 biochemical tests and possessed O-antigens characteristic of Providencia O-type strains 4 or 17. Study of the isolates indicated that endemic strains are capable of undergoing variation in urease activity. In the current classification urease-positive and urease-negative strains are classified as Proteus rettgeri and Providencia stuartii, respectively. The observed variation in urease activity of nosocomial isolates of Proteeae suggests that taxonomy should be modified so that all such strains would be accommodated in a single group.

The O-serotyping scheme for Providencia was tested on Providencia alcalifaciens isolates collected mostly from two hospitals. The specificites of the somatic (O) antigens of P. alcalifaciens were found to be different from those of Providencia stuartii, and separation of the Providencia typing scheme to allow separate typing of each species led to more efficient typing. All but 4 of 86 isolates were typable. Eighteen serotypes occurred among 53 typable isolates obtained from a pediatric hospital, and 11 occurred among 19 isolates from a general hospital. Thirty-two percent of the isolates from the pediatric hospital belonged to serotype O3, the most frequently isolated and most widely distributed type. The use of the serotyping scheme for P. alcalifaciens is advocated for further studies to examine strains of the species for enteropathogenic types.

A collection of 829 isolates of Providencia stuartii, mostly from urological specimens of patients in 12 hospitals, were O serotyped. Hospitals varied in serotype distribution, but most isolates (97%) fell into one or another of 14 O types of P. stuartii. One type (O63) was found in 10 hospitals, and six types (O4, O17, O25, O52, O55, O56) were found in 5 or more hospitals. These seven types were more common than others and included 753 (91%) of the isolates. Only four isolates agglutinated in Providencia alcalifaciens antisera and, for increased efficiency in serotyping, it is recommended that separate schemes be employed for P. stuartii and P. alcalifaciens. Strains endemic in different hospitals may differ in serotype and give rise to nosocomial infections that are clinically recognizable when infections occur in obvious clusters. Nosocomial infections occurring in low frequency among patients not located close to each other in the hospital may be detected with the aid of serotyping.

The somatic (O-) antigens of the type strains of the providencia antigenic scheme were examined for their biochemical reactions and their O-specificities. The scheme of 62 O-antigens was reconstituted from 52 original type strains and 10 strains substituted for originals that either were biochemically atypical of the genus or showed inappropriate serological reactions. Thirty-six type strains showed no significant relations with other type strains, and antisera could be used for typing without absorption. Among 26 type strains, significant reciprocal relations were demonstrated, and each cross-reacting antigen was examined for specificity and for its distribution among the type strains. Antisera to these strains required absorption with cell suspensions of other type strains for production of specificity in O-typing. Each typing antiserum, at low dilution, was shown to agglutinate homologous, but not heterologous, cell suspensions of type strains, and this result demonstrated the required specificity for typing on the basis of the O-antigens.