Chemotherapy-associated liver morphological changes in hepatic metastases (CALMCHeM).

PURPOSE: To review imaging findings in chemotherapy-associated liver morphological changes in hepatic metastases (CALMCHeM) on computed tomography (CT)/magnetic resonance imaging (MRI) and its association with tumor burden. METHODS: We performed a retrospective chart review to identify patients with hepatic metastases who received chemotherapy and subsequent follow-up imaging where CT or MRI showed morphological changes in the liver. The morphological changes searched for were nodularity, capsular retraction, hypodense fibrotic bands, lobulated outline, atrophy or hypertrophy of segments or lobes, widened fissures, and one or more features of portal hypertension (splenomegaly/venous collaterals/ascites). The inclusion criteria were as follows: a) no known chronic liver disease; b) availability of CT or MRI images before chemotherapy that showed no morphological signs of chronic liver disease; c) at least one follow-up CT or MRI image demonstrating CALMCHeM after chemotherapy. Two radiologists in consensus graded the initial hepatic metastases tumor burden according to number (≤10 and >10), lobe distribution (single or both lobes), and liver parenchyma volume affected (<50%, or ≥50%). Imaging features after treatment were graded according to a pre-defined qualitative assessment scale of "normal," "mild," "moderate," or "severe." Descriptive statistics were performed with binary groups based on the number, lobar distribution, type, and volume of the liver affected. Chi-square and t-tests were used for comparative statistics. The Cox proportional hazard model was used to determine the association between severe CALMCHeM changes and age, sex, tumor burden, and primary carcinoma type. RESULTS: A total of 219 patients met the inclusion criteria. The most common primaries were from breast (58.4%), colorectal (14.2%), and neuroendocrine (11.0%) carcinomas. Hepatic metastases were discrete in 54.8% of cases, confluent in 38.8%, and diffuse in 6.4%. The number of metastases was >10 in 64.4% of patients. The volume of liver involved was <50% in 79.8% and ≥50% in 20.2% of cases. The severity of CALMCHeM at the first imaging follow-up was associated with a larger number of metastases (P = 0.002) and volume of the liver affected (P = 0.015). The severity of CALMCHeM had progressed to moderate to severe changes in 85.9% of patients, and 72.5% of patients had one or more features of portal hypertension at the last follow-up. The most common features at the final follow-up were nodularity (95.0%), capsular retraction (93.4%), atrophy (66.2%), and ascites (65.7%). The Cox proportional hazard model showed metastases affected ≥50% of the liver (P = 0.033), and the female gender (P = 0.004) was independently associated with severe CALMCHeM. CONCLUSION: CALMCHeM can be observed with a wide variety of malignancies, is progressive in severity, and the severity correlates with the initial metastatic liver disease burden.

Light activation of Orange Carotenoid Protein reveals bicycle-pedal single-bond isomerization.

Orange Carotenoid protein (OCP) is the only known photoreceptor which uses carotenoid for its activation. It is found exclusively in cyanobacteria, where it functions to control light-harvesting of the photosynthetic machinery. However, the photochemical reactions and structural dynamics of this unique photosensing process are not yet resolved. We present time-resolved crystal structures at second-to-minute delays under bright illumination, capturing the early photoproduct and structures of the subsequent reaction intermediates. The first stable photoproduct shows concerted isomerization of C9'-C8' and C7'-C6' single bonds in the bicycle-pedal (s-BP) manner and structural changes in the N-terminal domain with minute timescale kinetics. These are followed by a thermally-driven recovery of the s-BP isomer to the dark state carotenoid configuration. Structural changes propagate to the C-terminal domain, resulting, at later time, in the H-bond rupture of the carotenoid keto group with protein residues. Solution FTIR and UV/Vis spectroscopy support the single bond isomerization of the carotenoid in the s-BP manner and subsequent thermal structural reactions as the basis of OCP photoreception.

On the earliest Acheulean in Britain: first dates and in-situ artefacts from the MIS 15 site of Fordwich (Kent, UK).

Northern Europe experienced cycles of hominin habitation and absence during the Middle Pleistocene. Fluvial gravel terrace sites in the east of Britain and north of France provide a majority of the data contributing to this understanding, mostly through the presence or absence of stone-tool artefacts. To date, however, relatively few sites have been radiometrically dated, and many have not been excavated in modern times, leading to an over-reliance on selectively sampled and poorly dated lithic assemblages. This includes Fordwich (Kent, UK), where over 330 bifaces were discovered through industrial quarrying in the 1920s. Here, we present the first excavation and dating of artefacts discovered in situ at Fordwich, alongside their technological analysis and relationship to those previously recovered. The site is demonstrated to retain deposits of Lower Palaeolithic artefacts, with 251 flakes, scrapers and cores identified to date. Infrared-radiofluorescence (IR-RF) dating of feldspar reveals 112 artefacts to have come from levels dating to at least 570 ± 36 to 513 ± 30 thousand years ago (ka) and are most plausibly assigned to an MIS 14 deposition, with artefacts produced during MIS 15 (approx. 560-620 ka). Indeed, these IR-RF samples provide minimum ages for artefacts. Combined with evidence from exposures linked to the original quarrying activities, a similar MIS 15 age is suggested for the more than 330 handaxe artefacts discovered in the 1920s. The remaining excavated artefacts come from levels dated to between 347 ± 22 and 385 ± 21 ka (MIS 10 or 11), with this later age interpreted to reflect post-MIS 14 deposition by substrate gullying and solifluction. These data demonstrate Fordwich to be one of the earliest Palaeolithic sites in northwestern Europe, and to retain the only large Acheulean handaxe assemblage directly dated to pre-MIS 13. Thus, Fordwich is determined to be a crucial piece of the pre-Anglian Palaeolithic puzzle in northern Europe.

Rapid and sensitive detection of SARS-CoV-2 infection using quantitative peptide enrichment LC-MS analysis.

Reliable, robust, large-scale molecular testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for monitoring the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a scalable analytical approach to detect viral proteins based on peptide immuno-affinity enrichment combined with liquid chromatography-mass spectrometry (LC-MS). This is a multiplexed strategy, based on targeted proteomics analysis and read-out by LC-MS, capable of precisely quantifying and confirming the presence of SARS-CoV-2 in phosphate-buffered saline (PBS) swab media from combined throat/nasopharynx/saliva samples. The results reveal that the levels of SARS-CoV-2 measured by LC-MS correlate well with their correspondingreal-time polymerase chain reaction (RT-PCR) read-out (r = 0.79). The analytical workflow shows similar turnaround times as regular RT-PCR instrumentation with a quantitative read-out of viral proteins corresponding to cycle thresholds (Ct) equivalents ranging from 21 to 34. Using RT-PCR as a reference, we demonstrate that the LC-MS-based method has 100% negative percent agreement (estimated specificity) and 95% positive percent agreement (estimated sensitivity) when analyzing clinical samples collected from asymptomatic individuals with a Ct within the limit of detection of the mass spectrometer (Ct ≤ 30). These results suggest that a scalable analytical method based on LC-MS has a place in future pandemic preparedness centers to complement current virus detection technologies.

The morphology of the Late Pleistocene hominin remains from the site of La Cotte de St Brelade, Jersey (Channel Islands).

Thirteen permanent fully erupted teeth were excavated at the Paleolithic site of La Cotte de St Brelade in Jersey in 1910 and 1911. These were all found in the same location, on a ledge behind a hearth in a Mousterian occupation level. They were originally identified as being Neanderthal. A fragment of occipital bone was found in a separate locality in a later season. Recent dating of adjacent sediments gives a probable age of <48 ka. The purpose of this article is to provide an updated description of the morphology of this material and consider its likely taxonomic assignment from comparison with Neanderthal and Homo sapiens samples. One of the original teeth has been lost, and we identify one as nonhominin. At least two adult individuals are represented. Cervix shape and the absence of common Neanderthal traits in several teeth suggest affinities with H. sapiens in both individuals, while crown and root dimensions and root morphology of all the teeth are entirely consistent with a Neanderthal attribution, pointing toward a possible shared Neanderthal and H. sapiens ancestry (the likely date of this material corresponds with the time in which both Neanderthals and H. sapiens were present in Europe). The occipital fragment is stratigraphically more recent and does not exhibit any diagnostic Neanderthal features.

An Improved Natural Transformation Protocol for the Cyanobacterium Synechocystis sp. PCC 6803.

The naturally transformable cyanobacterium Synechocystis sp. PCC 6803 is a widely used chassis strain for the photosynthetic production of chemicals. However, Synechocystis possesses multiple genome copies per cell which means that segregating mutations across all genome copies can be time-consuming. Here we use flow cytometry in combination with DNA staining to investigate the effect of phosphate deprivation on the genome copy number of the glucose-tolerant GT-P sub-strain of Synechocystis 6803. Like the PCC 6803 wild type strain, the ploidy of GT-P cells grown in BG-11 medium is growth phase dependent with an average genome copy number of 6.05 ± 0.27 in early growth (OD740 = 0.1) decreasing to 2.49 ± 0.11 in late stationary phase (OD740 = 7). We show that a 10-fold reduction in the initial phosphate concentration of the BG-11 growth medium reduces the average genome copy number of GT-P cells from 4.51 ± 0.20 to 2.94 ± 0.13 and increases the proportion of monoploid cells from 0 to 6% after 7 days of growth. In addition, we also show that the DnaA protein, which unusually for bacteria is not required for DNA replication in Synechocystis, plays a role in restoring polyploidy upon subsequent phosphate supplementation. Based on these observations, we have developed an alternative natural transformation protocol involving phosphate depletion that decreases the time required to obtain fully segregated mutants.

Precision multiparameter tracking of inflammation on timescales of hours to years using serial dried blood spots.

Aim: High-frequency longitudinal tracking of inflammation using dried blood microsamples provides a new window for personalized monitoring of infections, chronic inflammatory disease and clinical trials of anti-inflammatory drugs. Results/methodology: Using 1662 dried blood spot samples collected by 16 subjects over periods of weeks to years, we studied the behavior of 12 acute phase response and related proteins in inflammation events correlated with infection, vaccination, surgery, intense exercise and Crohn's disease. Proteins were measured using SISCAPA mass spectrometry and normalized to constant plasma volume using low-variance proteins, generating high precision within-person biomarker trajectories with well-characterized personal baselines. Discussion/conclusion: The results shed new light on the dynamic regulation of APR responses, offering a new approach to visualization of multidimensional inflammation trajectories.

Fluid Flow Patterns Through Drainage Catheters: Clinical Observations in 99 Patients.

PURPOSE: To describe patterns of fluid flow through locking pigtail and biliary catheters in patients that underwent biliary and abdominopelvic fluid drainage. METHODS: Contrast movement through catheter sideholes in pigtail and biliary catheters was evaluated retrospectively using sinograms and cholangiograms at 7-10 days post insertion. Dilute contrast injected through the catheter was evaluated by following flow through the catheter shaft and exit from side holes within the body cavity. Exit of contrast through side holes was appreciated and recorded. Included patients underwent biliary and abdominopelvic fluid drainage using 10.2-F catheters. Exclusion criteria included masking of contrast flow through sideholes by catheter angulation, contrast pooling or other imaging artifacts. RESULTS: A total of 99 patients meeting inclusion criteria underwent evaluation of contrast flow through pigtail (n = 81) and biliary (n = 18) catheters. For pigtail and biliary catheters, 91/99 cases (91.9%) showed contrast exiting the catheter from only the sidehole located most proximally to the catheter hub. In 6/99 cases (6.1%) contrast exited no further than the second most proximal sidehole. In 2/99 cases (2.0%) contrast exited no further than the third most proximal sidehole. In no cases was contrast observed exiting from distal sideholes beyond the third most proximal sidehole. CONCLUSION: Retrograde contrast injection through catheters suggests that the majority of the contribution to total output in drainage catheters comes from the most proximal side hole. Contribution of distal side holes to total drainage is negligible or non-existent, therefore the distal segment of the catheter may be considered non-functional.

Multiplexed longitudinal measurement of protein biomarkers in DBS using an automated SISCAPA workflow.

BACKGROUND: The use of DBS for quantitative protein biomarker measurement has been hindered by issues associated with blood hematocrit variations and lack of detection sensitivity, particularly when multiple biomarkers are measured. MATERIALS & METHODS: An automated, multiplexed SISCAPA analysis was used to normalize blood volume variations in DBS and quantify proteins of varying abundance in longitudinal specimens. CONCLUSION: The results showed that after normalizing the spot-to-spot hematocrit variations, peptide surrogates of protein biomarkers could be accurately quantitated in DBS. This allowed the establishment of baselines for a variety of biomarkers in multiple individuals and enabled detection of changes over time, thus offering an effective solution for longitudinal personal monitoring of biomarkers relevant in health and disease.

Characterization of Calflagin, a Flagellar Calcium-Binding Protein from Trypanosoma congolense.

BACKGROUND: Identification of species-specific trypanosome molecules is important for laboratory- and field-based research into epidemiology and disease diagnosis. Although Trypanosoma congolense is the most important trypanosome pathogen of cattle in Africa, no species-specific molecules found in infective bloodstream forms (BSF) of the parasites have been identified, thus limiting development of diagnostic tests. METHODS: Immuno-mass spectrometric methods were used to identify a protein that is recognized by a T. congolense-specific monoclonal antibody (mAb) Tc6/42.6.4. The identified molecule was expressed as a recombinant protein in E. coli and was tested in several immunoassays for its ability to interact with the mAb. The three dimensional structure of the protein was modeled and compared to crystal- and NMR-structures of the homologous proteins from T. cruzi and T. brucei respectively, in order to examine structural differences leading to the different immunoreactivity of the T. congolense molecule. Enzyme-linked immunosorbent assays (ELISA) were used to measure antibodies produced by trypanosome-infected African cattle in order to assess the potential for use of T. congolense calflagin in a serodiagnostic assay. RESULTS: The antigen recognized by the T. congolense-specific mAb Tc6/42.6.4 was identified as a flagellar calcium-binding protein, calflagin. The recombinant molecule showed immunoreactivity with the T. congolense-specific mAb confirming that it is the cognate antigen. Immunofluorescence experiments revealed that Ca2+ modulated the localization of the calflagin molecule in trypanosomes. Structural modelling and comparison with calflagin homologues from other trypanosomatids revealed four non-conserved regions on the surface of the T. congolense molecule that due to differences in surface chemistry and structural topography may form species-specific epitopes. ELISAs using the recombinant calflagin as antigen to detect antibodies in trypanosome-infected cattle showed that the majority of cattle had antibody responses. Area under the Receiver-Operating Characteristic (ROC) curves, associated with host IgG and IgM, were calculated to be 0.623 and 0.709 respectively, indicating a positive correlation between trypanosome infection and the presence of anti-calflagin antibodies. CONCLUSIONS: While calflagin is conserved among different species of African trypanosomes, our results show that T. congolense calflagin possesses unique epitopes that differentiate this protein from homologues in other trypanosome species. MAb Tc6/42.6.4 has clear utility as a laboratory tool for identifying T. congolense. T. congolense calflagin has potential as a serodiagnostic antigen and should be explored further for its utility in antigen-detection assays for diagnosis of cattle infections.

High precision quantification of human plasma proteins using the automated SISCAPA Immuno-MS workflow.

Efficient robotic workflows for trypsin digestion of human plasma and subsequent antibody-mediated peptide enrichment (the SISCAPA method) were developed with the goal of improving assay precision and throughput for multiplexed protein biomarker quantification. First, an 'addition only' tryptic digestion protocol was simplified from classical methods, eliminating the need for sample cleanup, while improving reproducibility, scalability and cost. Second, methods were developed to allow multiplexed enrichment and quantification of peptide surrogates of protein biomarkers representing a very broad range of concentrations and widely different molecular masses in human plasma. The total workflow coefficients of variation (including the 3 sequential steps of digestion, SISCAPA peptide enrichment and mass spectrometric analysis) for 5 proteotypic peptides measured in 6 replicates of each of 6 different samples repeated over 6 days averaged 3.4% within-run and 4.3% across all runs. An experiment to identify sources of variation in the workflow demonstrated that MRM measurement and tryptic digestion steps each had average CVs of ∼2.7%. Because of the high purity of the peptide analytes enriched by antibody capture, the liquid chromatography step is minimized and in some cases eliminated altogether, enabling throughput levels consistent with requirements of large biomarker and clinical studies.

Ethanol ablation in postbiopsy liver hemorrhage following two failed embolizations.

A 42-year-old male presented with intraperitoneal hemorrhage 5days following percutaneous liver biopsy for suspected hepatocellular carcinoma. Diagnostic angiogram localized the bleeding to segment VI hepatic artery branches. Two consecutive arterial embolizations with microspheres and platinum coils failed to control the bleeding. The patient was a poor surgical candidate, so ultrasound-guided ethanol ablation of the bleeding source and surrounding liver segment was employed as salvage therapy. The patient stabilized clinically and was discharged home to begin palliative therapy.

Effects of renal tubular dysfunction on bone in tenofovir-exposed HIV-positive patients.

OBJECTIVES: Tenofovir disoproxil fumarate (TDF) may cause renal tubular dysfunction (RTD) and reduce bone mineral density (BMD). We examined the relationship between RTD and BMD in TDF-exposed HIV-positive men. DESIGN AND METHODS: We analysed urinary retinol-binding protein/creatinine ratio (RBPCR) and fractional excretion of phosphate (FEPO4) to quantify RTD in a cross-sectional sample of randomly selected HIV-positive men at a single tertiary outpatient clinic. BMD at the lumbar spine and hip was measured by dual-energy X-ray absorptiometry. Multivariate logistic regression was used to analyse factors associated with RTD, and linear regression to examine the relationship between RTD and BMD. RESULTS: Of 293 men (mean age 48 years, 94% White ethnicity, median TDF exposure 2.1 years), 22.5% had RBPCR-defined RTD and 12.3% had FEPO4-defined RTD. We observed a negative correlation between RBPCR and BMD at the spine (ß -0.2, P = 0.002) and hip (total: ß -0.1, P = 0.02; femoral neck: ß -0.1, P = 0.02), but not between FePO4 and BMD. In multivariable analyses, RTD defined by more than five-fold elevations in RBPCR was associated with significantly lower BMD of the spine. CONCLUSION: In HIV-positive patients receiving TDF-containing antiretroviral therapy, RTD was associated with lower BMD of the spine in HIV-positive men. RBPCR quantification may identify patients at increased risk of TDF-associated BMD loss.

O-Methyltransferase is shared between the pentose phosphate and shikimate pathways and is essential for mycosporine-like amino acid biosynthesis in Anabaena variabilis ATCC 29413.

The parent core structure of mycosporine-like amino acids (MAAs) is 4-deoxygadusol, which, in cyanobacteria, is derived from conversion of the pentose phosphate pathway intermediate sedoheptulose 7-phosphate by the enzymes 2-epi-5-epivaliolone synthase (EVS) and O-methyltransferase (OMT). Yet, deletion of the EVS gene from Anabaena variabilis ATCC 29413 was shown to have little effect on MAA production, thus suggesting that its biosynthesis is not exclusive to the pentose phosphate pathway. Herein, we report how, using pathway-specific inhibitors, we demonstrated unequivocally that MAA biosynthesis occurs also via the shikimate pathway. In addition, complete in-frame gene deletion of the OMT gene from A. variabilis ATCC 29413 reveals that, although biochemically distinct, the pentose phosphate and shikimate pathways are inextricably linked to MAA biosynthesis in this cyanobacterium. Furthermore, proteomic data reveal that the shikimate pathway is the predominate route for UV-induced MAA biosynthesis.

High-throughput SISCAPA quantitation of peptides from human plasma digests by ultrafast, liquid chromatography-free mass spectrometry.

We investigated the utility of an SPE-MS/MS platform in combination with a modified SISCAPA workflow for chromatography-free MRM analysis of proteotypic peptides in digested human plasma. This combination of SISCAPA and SPE-MS/MS technology allows sensitive, MRM-based quantification of peptides from plasma digests with a sample cycle time of ∼7 s, a 300-fold improvement over typical MRM analyses with analysis times of 30-40 min that use liquid chromatography upstream of MS. The optimized system includes capture and enrichment to near purity of target proteotypic peptides using rigorously selected, high affinity, antipeptide monoclonal antibodies and reduction of background peptides using a novel treatment of magnetic bead immunoadsorbents. Using this method, we have successfully quantitated LPS-binding protein and mesothelin (concentrations of ∼5000 ng/mL and ∼10 ng/mL, respectively) in human plasma. The method eliminates the need for upstream liquid-chromatography and can be multiplexed, thus facilitating quantitative analysis of proteins, including biomarkers, in large sample sets. The method is ideal for high-throughput biomarker validation after affinity enrichment and has the potential for applications in clinical laboratories.

Interlaboratory evaluation of automated, multiplexed peptide immunoaffinity enrichment coupled to multiple reaction monitoring mass spectrometry for quantifying proteins in plasma.

The inability to quantify large numbers of proteins in tissues and biofluids with high precision, sensitivity, and throughput is a major bottleneck in biomarker studies. We previously demonstrated that coupling immunoaffinity enrichment using anti-peptide antibodies (SISCAPA) to multiple reaction monitoring mass spectrometry (MRM-MS) produces Immunoprecipitation MRM-MS (immuno-MRM-MS) assays that can be multiplexed to quantify proteins in plasma with high sensitivity, specificity, and precision. Here we report the first systematic evaluation of the interlaboratory performance of multiplexed (8-plex) immuno-MRM-MS in three independent labs. A staged study was carried out in which the effect of each processing and analysis step on assay coefficient of variance, limit of detection, limit of quantification, and recovery was evaluated. Limits of detection were at or below 1 ng/ml for the assayed proteins in 30 µl of plasma. Assay reproducibility was acceptable for verification studies, with median intra- and interlaboratory coefficients of variance above the limit of quantification of 11% and <14%, respectively, for the entire immuno-MRM-MS assay process, including enzymatic digestion of plasma. Trypsin digestion and its requisite sample handling contributed the most to assay variability and reduced the recovery of target peptides from digested proteins. Using a stable isotope-labeled protein as an internal standard instead of stable isotope-labeled peptides to account for losses in the digestion process nearly doubled assay accuracy for this while improving assay precision 5%. Our results demonstrate that multiplexed immuno-MRM-MS can be made reproducible across independent laboratories and has the potential to be adopted widely for assaying proteins in matrices as complex as plasma.

Quantification of proteins using peptide immunoaffinity enrichment coupled with mass spectrometry.

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').(1) An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).(2) In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules (3, 4) and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.(5-7) To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.(8-13) In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Evaluation of large scale quantitative proteomic assay development using peptide affinity-based mass spectrometry.

Stable isotope standards and capture by antipeptide antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution and detection by multiple reaction monitoring mass spectrometry to provide quantitative measurement of peptides as surrogates for their respective proteins. In this report, we describe a feasibility study to determine the success rate for production of suitable antibodies for SISCAPA assays in order to inform strategies for large-scale assay development. A workflow was designed that included a multiplex immunization strategy in which up to five proteotypic peptides from a single protein target were used to immunize individual rabbits. A total of 403 proteotypic tryptic peptides representing 89 protein targets were used as immunogens. Antipeptide antibody titers were measured by ELISA and 220 antipeptide antibodies representing 89 proteins were chosen for affinity purification. These antibodies were characterized with respect to their performance in SISCAPA-multiple reaction monitoring assays using trypsin-digested human plasma matrix. More than half of the assays generated were capable of detecting the target peptide at concentrations of less than 0.5 fmol/µl in human plasma, corresponding to protein concentrations of less than 100 ng/ml. The strategy of multiplexing five peptide immunogens was successful in generating a working assay for 100% of the targeted proteins in this evaluation study. These results indicate it is feasible for a single laboratory to develop hundreds of assays per year and allow planning for cost-effective generation of SISCAPA assays.

Foliage chemistry influences tree choice and landscape use of a gliding marsupial folivore.

The chemical quality of forage may determine landscape use and habitat quality for some herbivorous species. However, studies that investigate the relationship between foliar chemistry and foraging choices in wild vertebrates are rare. Petauroides volans (the greater glider) is unique among Australian marsupial folivores because it glides. It also frequently consumes foliage from both major Eucalyptus subgenera, Eucalyptus (common name "monocalypt") and Symphyomyrtus (common name "symphyomyrtle"), which differ markedly in their foliar chemistry. Such differences are thought to be a product of co-evolution that also led to guild-specific plant secondary metabolite (PSM) specialization among other marsupial eucalypt folivores. To explore whether foliar chemistry influences tree use, we analyzed foliage from eucalypt trees in which we observed P. volans during a radio tracking study and from eucalypt trees in which animals were never observed. We used a combination of chemical assays and near infrared spectrophotometry (NIRS) to determine concentrations of nitrogen (N), in vitro available nitrogen (AvailN), and in vitro digestible dry matter (DDM) from foliage sampled from the monocalypt and symphyomyrtle species, and total formylated phloroglucinol compounds (FPCs) and sideroxylonals (a class of FPCs) from the symphyomyrtle species (FPCs do not occur in monocalypts). Tree size and spatially-dependent, intraspecific variations in sideroxylonals and DDM concentrations in the symphyomyrtle foliage and of N, AvailN, and DDM in the monocalypt species were important indicators of tree use and habitat suitability for P. volans. The results i) demonstrate that guild-specific PSMs do not always lead to guild-specific foraging; ii) provide a compelling co-evolutionary case for the development of gliding in P. volans; and iii) have implications for the management and conservation of this and other folivorous species.

MALDI immunoscreening (MiSCREEN): a method for selection of anti-peptide monoclonal antibodies for use in immunoproteomics.

A scalable method for screening and selection of peptide-specific monoclonal antibodies (mAbs) is described. To identify high affinity anti-peptide mAbs in hybridoma supernatants, antibodies were captured by magnetic affinity beads followed by binding of specific peptides from solution. After timed washing steps, the remaining bound peptides were eluted from the beads and detected by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). This allowed measurement of monovalent interactions of peptides with single antigen binding sites on the antibodies, thus reflecting antibody affinity rather than avidity. Antibodies that were able to bind target peptides from solution phase and retain them during washing for a minimum of 10 min were identified by the strength of the appropriate m/z peptide MS signals obtained. This wash time reflects the minimum peptide dissociation time required for use of these antibodies in several current immuno-mass spectrometry assays. Kinetic analysis of antibody-peptide binding by surface plasmon resonance (SPR) showed that the selected antibodies were of high affinity and, most importantly, had low dissociation constants. This method, called MALDI immunoscreening (MiSCREEN), thus enables rapid screening and selection of high affinity anti-peptide antibodies that are useful for a variety of immunoproteomics applications. To demonstrate their functional utility in immuno-mass spectrometry assays, we used the selected, purified RabMAbs to enrich natural (tryptic) peptides from digested human plasma.