Detection of drug resistant Mycobacterium tuberculosis by high-throughput sequencing of DNA isolated from acid fast bacilli smears.

BACKGROUND: Drug susceptibility testing for Mycobacterium tuberculosis (MTB) is difficult to perform in resource-limited settings where Acid Fast Bacilli (AFB) smears are commonly used for disease diagnosis and monitoring. We developed a simple method for extraction of MTB DNA from AFB smears for sequencing-based detection of mutations associated with resistance to all first and several second-line anti-tuberculosis drugs. METHODS: We isolated MTB DNA by boiling smear content in a Chelex solution, followed by column purification. We sequenced PCR-amplified segments of the rpoB, katG, embB, gyrA, gyrB, rpsL, and rrs genes, the inhA, eis, and pncA promoters and the entire pncA gene. RESULTS: We tested our assay on 1,208 clinically obtained AFB smears from Ghana (n = 379), Kenya (n = 517), Uganda (n = 262), and Zambia (n = 50). Coverage depth varied by target and slide smear grade, ranging from 300X to 12000X on average. Coverage of ≥20X was obtained for all targets in 870 (72%) slides overall. Mono-resistance (5.9%), multi-drug resistance (1.8%), and poly-resistance (2.4%) mutation profiles were detected in 10% of slides overall, and in over 32% of retreatment and follow-up cases. CONCLUSION: This rapid AFB smear DNA-based method for determining drug resistance may be useful for the diagnosis and surveillance of drug-resistant tuberculosis.

A herpesvirus transactivator and cellular POU proteins extensively regulate DNA binding of the host Notch signaling protein RBP-Jκ to the virus genome.

Reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) from latency requires the viral transactivator Rta to contact the host protein Jκ recombination signal-binding protein (RBP-Jκ or CSL). RBP-Jκ normally binds DNA sequence-specifically to determine the transcriptional targets of the Notch-signaling pathway, yet Notch alone cannot reactivate KSHV. We previously showed that Rta stimulates RBP-Jκ DNA binding to the viral genome. On a model viral promoter, this function requires Rta to bind to multiple copies of an Rta DNA motif (called "CANT" or Rta-c) proximal to an RBP-Jκ motif. Here, high-resolution ChIP/deep sequencing from infected primary effusion lymphoma cells revealed that RBP-Jκ binds nearly exclusively to different sets of viral genome sites during latency and reactivation. RBP-Jκ bound DNA frequently, but not exclusively, proximal to Rta bound to single, but not multiple, Rta-c motifs. To discover additional regulators of RBP-Jκ DNA binding, we used bioinformatics to identify cellular DNA-binding protein motifs adjacent to either latent or reactivation-specific RBP-Jκ-binding sites. Many of these cellular factors, including POU class homeobox (POU) proteins, have known Notch or herpesvirus phenotypes. Among a set of Rta- and RBP-Jκ-bound promoters, Rta transactivated only those that also contained POU motifs in conserved positions. On some promoters, POU factors appeared to inhibit RBP-Jκ DNA binding unless Rta bound to a proximal Rta-c motif. Moreover, POU2F1/Oct-1 expression was induced during KSHV reactivation, and POU2F1 knockdown diminished infectious virus production. Our results suggest that Rta and POU proteins broadly regulate DNA binding of RBP-Jκ during KSHV reactivation.

"Tuberculosis in advanced HIV infection is associated with increased expression of IFNγ and its downstream targets".

BACKGROUND: Tuberculosis (TB) is the major cause of death in Human Immunodeficiency Virus (HIV)-infected individuals. However, diagnosis of TB in HIV remains challenging particularly when HIV infection is advanced. Several gene signatures and serum protein biomarkers have been identified that distinguish active TB from latent infection. Our study was designed to assess if gene expression signatures and cytokine levels would distinguish active TB in advanced HIV. METHODS: We conducted a case-control study of whole blood RNA-Seq and plasma cytokine/chemokine analysis in HIV-infected with CD4+ T cell count of ≤ 100 cells/µl, with and without active TB. Next, the overlap of the differentially expressed genes (DEG) with the published signatures was performed and then receiver operator characteristic (ROC) analysis was done on small gene discriminators to determine their performance in distinguishing TB in advanced HIV. ELISA was performed on plasma to evaluate cytokine and chemokine levels. RESULTS: Hierarchical clustering of the transcriptional profiles showed that, in general, HIV-infected individuals with TB (TB-HIV) clustered separately from those without TB. IPA indicated that the TB-HIV signature was characterized by an increase in inflammatory signaling pathways. Analysis of overlaps between DEG in our data set with published TB signatures revealed that significant overlap was seen with one TB signature and one TB-IRIS signature. ROC analysis revealed that transcript levels of FcGR1A (AUC = 0.85) and BATF2 (AUC = 0.82), previously reported as consistent single gene classifiers of active TB irrespective of HIV status, performed successfully even in advanced HIV. Plasma protein levels of IFNγ, a stimulator of FcGR1A and BATF2, and CXCL10, also up-regulated by IFNγ, accurately classified active TB (AUC = 0.98 and 0.91, respectively) in advanced HIV. Neither of these genes nor proteins distinguished between TB and TB-IRIS. CONCLUSIONS: Gene expression of FcGR1A and BATF2, and plasma protein levels of IFNγ and CXCL10 have the potential to independently detect TB in advanced HIV. However, since other lung diseases were not included in this study, these final candidates need to be validated as specific to TB in the advanced HIV population with TB.

The human GCOM1 complex gene interacts with the NMDA receptor and internexin-alpha.

The known functions of the human GCOM1 complex hub gene include transcription elongation and the intercalated disk of cardiac myocytes. However, in all likelihood, the gene's most interesting, and thus far least understood, roles will be found in the central nervous system. To investigate the functions of the GCOM1 gene in the CNS, we have cloned human and rat brain cDNAs encoding novel, 105 kDa GCOM1 combined (Gcom) proteins, designated Gcom15, and identified a new group of GCOM1 interacting genes, termed Gints, from yeast two-hybrid (Y2H) screens. We showed that Gcom15 interacts with the NR1 subunit of the NMDA receptor by co-expression in heterologous cells, in which we observed bi-directional co-immunoprecipitation of human Gcom15 and murine NR1. Our Y2H screens revealed 27 novel GCOM1 interacting genes, many of which are synaptic proteins and/or play roles in neurologic diseases. Finally, we showed, using rat brain protein preparations, that the Gint internexin-alpha (INA), a known interactor of the NMDAR, co-IPs with GCOM1 proteins, suggesting a GCOM1-GRIN1-INA interaction and a novel pathway that may be relevant to neuroprotection.

Type III interferon is a critical regulator of innate antifungal immunity.

Type III interferons (IFN-λs) are the most recently found members of the IFN cytokine family and engage IFNLR1 and IL10R2 receptor subunits to activate innate responses against viruses. We have identified IFN-λs as critical instructors of antifungal neutrophil responses. Using Aspergillus fumigatus (Af) as a model to study antifungal immune responses, we found that depletion of CCR2+ monocytes compromised the ability of neutrophils to control invasive fungal growth. Using an unbiased approach, we identified type I and III IFNs as critical regulators of the interplay between monocytes and neutrophils responding to Af We found that CCR2+ monocytes are an important early source of type I IFNs that prime optimal expression of IFN-λ. Type III IFNs act directly on neutrophils to activate their antifungal response, and mice with neutrophil-specific deletion of IFNLR1 succumb to invasive aspergillosis. Dysfunctional neutrophil responses in CCR2-depleted mice were rescued by adoptive transfer of pulmonary CCR2+ monocytes or by exogenous administration of IFN-α and IFN-λ. Thus, CCR2+ monocytes promote optimal activation of antifungal neutrophils by initiating a coordinated IFN response. We have identified type III IFNs as critical regulators of neutrophil activation and type I IFNs as early stimulators of IFN-λ expression.

Tudor-SN-mediated endonucleolytic decay of human cell microRNAs promotes G1/S phase transition.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression. The pathways that mediate mature miRNA decay are less well understood than those that mediate miRNA biogenesis. We found that functional miRNAs are degraded in human cells by the endonuclease Tudor-SN (TSN). In vitro, recombinant TSN initiated the decay of both protein-free and Argonaute 2-loaded miRNAs via endonucleolytic cleavage at CA and UA dinucleotides, preferentially at scissile bonds located more than five nucleotides away from miRNA ends. Cellular targets of TSN-mediated decay defined using microRNA sequencing followed this rule. Inhibiting TSN-mediated miRNA decay by CRISPR-Cas9 knockout of TSN inhibited cell cycle progression by up-regulating a cohort of miRNAs that down-regulates mRNAs that encode proteins critical for the G1-to-S phase transition. Our study indicates that targeting TSN nuclease activity could inhibit pathological cell proliferation.

Cutting Edge: Helminth Coinfection Blocks Effector Differentiation of CD8 T Cells through Alternate Host Th2- and IL-10-Mediated Responses.

Concurrent helminth infection potently inhibits T cell immunity; however, whether helminthes prevent T cell priming or skew clonal recruitment and effector differentiation is not known. Using coinfection with two natural mouse pathogens, Heligmosomoides polygyrus and Toxoplasma gondii, to investigate the negative impact of helminthes on the CD8 T cell response, we demonstrate helminth-induced suppression of IL-12-dependent differentiation of killer-like receptor G1+ effector CD8 T cells and IFN-γ production. Nevertheless, reversal of helminth suppression of the innate IL-12 response of CD8α+ dendritic cells, which occurred in STAT6-deficient mice, was not sufficient to normalize CD8 T cell differentiation. Instead, a combined deficiency in IL-4 and IL-10 was required to reverse the negative effects of helminth coinfection on the CD8 T cell response. Monoclonal T. gondii-specific CD8 T cells adoptively transferred into coinfected mice recapitulated the spectrum of helminth-induced effects on the polyclonal CD8 T response, indicating the lack of requirement for clonal skewing.

Transcriptional and Physiological Changes during Mycobacterium tuberculosis Reactivation from Non-replicating Persistence.

Mycobacterium tuberculosis can persist for years in the hostile environment of the host in a non-replicating or slowly replicating state. While active disease predominantly results from reactivation of a latent infection, the molecular mechanisms of M. tuberculosis reactivation are still poorly understood. We characterized the physiology and global transcriptomic profiles of M. tuberculosis during reactivation from hypoxia-induced non-replicating persistence. We found that M. tuberculosis reactivation upon reaeration was associated with a lag phase, in which the recovery of cellular physiological and metabolic functions preceded the resumption of cell replication. Enrichment analysis of the transcriptomic dynamics revealed changes to many metabolic pathways and transcription regulons/subnetworks that orchestrated the metabolic and physiological transformation in preparation for cell division. In particular, we found that M. tuberculosis reaeration lag phase is associated with down-regulation of persistence-associated regulons/subnetworks, including DosR, MprA, SigH, SigE, and ClgR, as well as metabolic pathways including those involved in the uptake of lipids and their catabolism. More importantly, we identified a number of up-regulated transcription regulons and metabolic pathways, including those involved in metal transport and remobilization, second messenger-mediated responses, DNA repair and recombination, and synthesis of major cell wall components. We also found that inactivation of the major alternative sigma factors SigE or SigH disrupted exit from persistence, underscoring the importance of the global transcriptional reprogramming during M. tuberculosis reactivation. Our observations suggest that M. tuberculosis lag phase is associated with a global gene expression reprogramming that defines the initiation of a reactivation process.

Dorsal root ganglion transcriptome analysis following peripheral nerve injury in mice.

BACKGROUND: Peripheral nerve injury leads to changes in gene expression in primary sensory neurons of the injured dorsal root ganglia. These changes are believed to be involved in neuropathic pain genesis. Previously, these changes have been identified using gene microarrays or next generation RNA sequencing with poly-A tail selection, but these approaches cannot provide a more thorough analysis of gene expression alterations after nerve injury. METHODS: The present study chose to eliminate mRNA poly-A tail selection and perform strand-specific next generation RNA sequencing to analyze whole transcriptomes in the injured dorsal root ganglia following spinal nerve ligation. Quantitative real-time reverse transcriptase polymerase chain reaction assay was carried out to verify the changes of some differentially expressed RNAs in the injured dorsal root ganglia after spinal nerve ligation. RESULTS: Our results showed that more than 50 million (M) paired mapped sequences with strand information were yielded in each group (51.87 M-56.12 M in sham vs. 51.08 M-57.99 M in spinal nerve ligation). Six days after spinal nerve ligation, expression levels of 11,163 out of a total of 27,463 identified genes in the injured dorsal root ganglia significantly changed, of which 52.14% were upregulated and 47.86% downregulated. The largest transcriptional changes were observed in protein-coding genes (91.5%) followed by noncoding RNAs. Within 944 differentially expressed noncoding RNAs, the most significant changes were seen in long interspersed noncoding RNAs followed by antisense RNAs, processed transcripts, and pseudogenes. We observed a notable proportion of reads aligning to intronic regions in both groups (44.0% in sham vs. 49.6% in spinal nerve ligation). Using quantitative real-time polymerase chain reaction, we confirmed consistent differential expression of selected genes including Kcna2, Oprm1 as well as lncRNAs Gm21781 and 4732491K20Rik following spinal nerve ligation. CONCLUSION: Our findings suggest that next generation RNA sequencing can be used as a promising approach to analyze the changes of whole transcriptomes in dorsal root ganglia following nerve injury and to possibly identify new targets for prevention and treatment of neuropathic pain.

Proteomic analysis of oral cavity squamous cell carcinoma specimens identifies patient outcome-associated proteins.

CONTEXT: Global proteomic analysis of oral cavity squamous cell carcinoma was performed to identify changes that reflect patient outcomes. OBJECTIVES: To identify differentially expressed proteins associated with patient outcomes and to explore the use of imaging mass spectrometry as a clinical tool to identify clinically relevant proteins. DESIGN: Two-dimensional separation of digested peptides generated from 43 specimens with high-resolution mass spectrometry identified proteins associated with disease-specific death, distant metastasis, and loco-regional recurrence. RNA expressions had been correlated to protein levels to test transcriptional regulation of clinically relevant proteins. Imaging mass spectrometry explored an alternative platform for assessing clinically relevant proteins that would complement surgical pathologic diagnosis. RESULTS: Seventy-two peptide features were found to be associated with 3 patient outcomes: disease-specific death (9), distant metastasis (16), and loco-regional recurrence (39); 8 of them were associated with multiple outcomes. Functional ontology revealed major changes in cell adhesion and calcium binding. Thirteen RNAs showed strong correlation with their encoded proteins, implying transcriptional control. Reduction of DSP, PKP1, and TRIM29 was associated with significantly shorter time to onset of distant metastasis. Reduction of PKP1 and TRIM29 correlated with poorer disease-specific survival. Additionally, S100A8 and S100A9 reductions were verified for their association with poor prognosis using imaging mass spectrometry, a platform more adaptable for use with surgical pathology. CONCLUSIONS: Using global proteomic analysis, we have identified proteins associated with clinical outcomes. The list of clinically relevant proteins observed will provide a means to develop clinical assays for prognosis and optimizing treatment selection.

MIRD pamphlet No. 25: MIRDcell V2.0 software tool for dosimetric analysis of biologic response of multicellular populations.

Patients undergoing nuclear medicine procedures for cancer therapy are administered radiopharmaceuticals that emit various types of radiation. Because radiation has differential delivery to and uptake by cells in tissue, radiation exposures are often highly nonuniform. Some cell populations in a tissue may contain widely different amounts of radioactivity, whereas other cell populations in the same tissue may contain no radioactivity, referred to as labeled and unlabeled cells, respectively. Furthermore, the toxicity of the radiations emitted can depend on the location of the radioactive decay within the cell (e.g., nucleus vs. cytoplasm). Therefore, the response of a given cell depends on the absorbed dose received from radiations emitted by decays within the cell (self-dose) and emitted by decays in neighboring cells (cross-dose), among other factors. Taken together, these variables make it difficult to predict the response of cell populations to radiopharmaceuticals. Accordingly, to assist in designing treatment plans for therapeutic radiopharmaceuticals, an applet software application called MIRDcell was developed. This applet models the distribution of radiopharmaceuticals in tissues, calculates the distribution of radiation dose, models responses on a cell-by-cell basis, and predicts the surviving fraction of the labeled and unlabeled cell populations. MIRDcell can be accessed at http://mirdcell.njms.rutgers.edu/.

Inflammatory monocytes orchestrate innate antifungal immunity in the lung.

Aspergillus fumigatus is an environmental fungus that causes invasive aspergillosis (IA) in immunocompromised patients. Although -CC-chemokine receptor-2 (CCR2) and Ly6C-expressing inflammatory monocytes (CCR2⁺Mo) and their derivatives initiate adaptive pulmonary immune responses, their role in coordinating innate immune responses in the lung remain poorly defined. Using conditional and antibody-mediated cell ablation strategies, we found that CCR2⁺Mo and monocyte-derived dendritic cells (Mo-DCs) are essential for innate defense against inhaled conidia. By harnessing fluorescent Aspergillus reporter (FLARE) conidia that report fungal cell association and viability in vivo, we identify two mechanisms by which CCR2⁺Mo and Mo-DCs exert innate antifungal activity. First, CCR2⁺Mo and Mo-DCs condition the lung inflammatory milieu to augment neutrophil conidiacidal activity. Second, conidial uptake by CCR2⁺Mo temporally coincided with their differentiation into Mo-DCs, a process that resulted in direct conidial killing. Our findings illustrate both indirect and direct functions for CCR2⁺Mo and their derivatives in innate antifungal immunity in the lung.

Exome sequencing in familial corticobasal degeneration.

BACKGROUND: Corticobasal degeneration (CBD) is a neurodegenerative, sporadic disorder of unknown cause. Few familial cases have been described. OBJECTIVE: We aim to characterize the clinical, imaging, pathological and genetic features of two familial cases of CBD. METHODS: We describe two first cousins with CBD associated with atypical MRI findings. We performed exome sequencing in both subjects and in an unaffected first cousin of similar age. RESULTS: The cases include a 79-year-old woman and a 72-year-old man of Native American and British origin. The onset of the neurological manifestations was 74 and 68 years respectively. Both patients presented with a combination of asymmetric parkinsonism, apraxia, myoclonic tremor, cortical sensory syndrome, and gait disturbance. The female subject developed left side fixed dystonia. The manifestations were unresponsive to high doses of levodopa in both cases. Extensive bilateral T1-W hyperintensities and T2-W hypointensities in basal ganglia and thalamus were observed in the female patient; whereas these findings were more subtle in the male subject. Postmortem examination of both patients was consistent with corticobasal degeneration; the female patient had additional findings consistent with mild Alzheimer's disease. No Lewy bodies were found in either case. Exome sequencing showed mutations leading to possible structural changes in MRS2 and ZHX2 genes, which appear to have the same upstream regulator miR-4277. CONCLUSIONS: Corticobasal degeneration can have a familial presentation; the role of MRS2 and ZHX2 gene products in CBD should be further investigated.

RNA-Seq for enrichment and analysis of IRF5 transcript expression in SLE.

Polymorphisms in the interferon regulatory factor 5 (IRF5) gene have been consistently replicated and shown to confer risk for or protection from the development of systemic lupus erythematosus (SLE). IRF5 expression is significantly upregulated in SLE patients and upregulation associates with IRF5-SLE risk haplotypes. IRF5 alternative splicing has also been shown to be elevated in SLE patients. Given that human IRF5 exists as multiple alternatively spliced transcripts with distinct function(s), it is important to determine whether the IRF5 transcript profile expressed in healthy donor immune cells is different from that expressed in SLE patients. Moreover, it is not currently known whether an IRF5-SLE risk haplotype defines the profile of IRF5 transcripts expressed. Using standard molecular cloning techniques, we identified and isolated 14 new differentially spliced IRF5 transcript variants from purified monocytes of healthy donors and SLE patients to generate an IRF5 variant transcriptome. Next-generation sequencing was then used to perform in-depth and quantitative analysis of full-length IRF5 transcript expression in primary immune cells of SLE patients and healthy donors by next-generation sequencing. Evidence for additional alternatively spliced transcripts was obtained from de novo junction discovery. Data from these studies support the overall complexity of IRF5 alternative splicing in SLE. Results from next-generation sequencing correlated with cloning and gave similar abundance rankings in SLE patients thus supporting the use of this new technology for in-depth single gene transcript profiling. Results from this study provide the first proof that 1) SLE patients express an IRF5 transcript signature that is distinct from healthy donors, 2) an IRF5-SLE risk haplotype defines the top four most abundant IRF5 transcripts expressed in SLE patients, and 3) an IRF5 transcript signature enables clustering of SLE patients with the H2 risk haplotype.

Two-dimensional method for time aligning liquid chromatography-mass spectrometry data.

We describe a new time alignment method that takes advantage of both dimensions of LC-MS data to resolve ambiguities in peak matching while remaining computationally efficient. This approach, Warp2D, combines peak extraction with a two-dimensional correlation function to provide a reliable alignment scoring function that is insensitive to spurious peaks and background noise. One-dimensional alignment methods are often based on the total-ion-current elution profile of the spectrum and are unable to distinguish peaks of different masses. Our approach uses one-dimensional alignment in time, but with a scoring function derived from the overlap of peaks in two dimensions, thereby combining the specificity of two-dimensional methods with the computational performance of one-dimensional methods. The peaks are approximated as two-dimensional Gaussians of varying width. This approximation allows peak overlap (the measure of alignment quality) to be calculated analytically, without computationally intensive numerical integration in two dimensions. To demonstrate the general applicability of Warp2D, we chose a variety of complex samples that have substantial biological and analytical variability, including human serum and urine. We show that Warp2D works well with these diverse sample sets and with minimal tuning of parameters, based on the reduced standard deviation of peak elution times after warping. The combination of high computational speed, robustness with complex samples, and lack of need for detailed tuning makes this alignment method well suited to high-throughput LC-MS studies.

A noise model for mass spectrometry based proteomics.

MOTIVATION: Mass spectrometry data are subjected to considerable noise. Good noise models are required for proper detection and quantification of peptides. We have characterized noise in both quadrupole time-of-flight (Q-TOF) and ion trap data, and have constructed models for the noise. RESULTS: We find that the noise in Q-TOF data from Applied Biosystems QSTAR fits well to a combination of multinomial and Poisson model with detector dead-time correction. In comparison, ion trap noise from Agilent MSD-Trap-SL is larger than the Q-TOF noise and is proportional to Poisson noise. We then demonstrate that the noise model can be used to improve deisotoping for peptide detection, by estimating appropriate cutoffs of the goodness of fit parameter at prescribed error rates. The noise models also have implications in noise reduction, retention time alignment and significance testing for biomarker discovery.

Global proteomic analysis distinguishes biologic differences in head and neck squamous carcinoma.

The goal of this study was to establish a method for detecting biologically significant differences in protein expression of head and neck squamous cell carcinoma (HNSCC) obtained from the same samples utilized in gene expression analyses. Proteins from two head and neck tumor cell lines, SCC-25 and FaDu, were isolated from the denatured protein solution remaining from the TRIzol extraction procedure used for isolation of total RNA for microarray analysis. Peptides resulting from chemical and enzymatic digestion of the proteins were first separated by strong cation-exchange chromatography, followed by liquid chromatography-mass spectrometry (LC-MS) analysis on a QqTOF mass spectrometer. Stable isotope-labeled synthetic peptides were added to each ion-exchange fraction as internal standards, for reversed-phase HPLC retention time alignment. Protein extraction and digestion were repeated three times for each cell line and each extract was analyzed three times by LC-MS. To discriminate between technical vs biological variation, the ion-exchange fraction, retention time, normalized mass and signal intensity of these nine data sets were constructed into numerical arrays for statistical analysis. Of the approximately 50,000 signals, 90 peptide ions were found to discriminate the two cell lines with high stringency. Of those, six peptides were derived from vimentin and four peptides were derived from annexin II; both expressed more in SCC-25. Follow-up analysis of some of these signals by LC-MS/MS and RNA expression profiling revealed both concordance and discordance of RNA and protein expression. This study demonstrates that this procedure is highly reliable for identifying peptides that distinguish biological variability among samples, indicating that this method can be applied to study clinical samples, to identify potential prognostic biomarkers for HNSCC.

Data reduction of isotope-resolved LC-MS spectra.

MOTIVATION: Data reduction of liquid chromatography-mass spectrometry (LC-MS) spectra can be a challenge due to the inherent complexity of biological samples, noise and non-flat baseline. We present a new algorithm, LCMS-2D, for reliable data reduction of LC-MS proteomics data. RESULTS: LCMS-2D can reliably reduce LC-MS spectra with multiple scans to a list of elution peaks, and subsequently to a list of peptide masses. It is capable of noise removal, and deconvoluting peaks that overlap in m/z, in retention time, or both, by using a novel iterative peak-picking step, a 'rescue' step, and a modified variable selection method. LCMS-2D performs well with three sets of annotated LC-MS spectra, yielding results that are better than those from PepList, msInspect and the vendor software BioAnalyst. AVAILABILITY: The software LCMS-2D is available under the GNU general public license from http://www.bioc.aecom.yu.edu/labs/angellab/as a standalone C program running on LINUX.

Automatic deconvolution of isotope-resolved mass spectra using variable selection and quantized peptide mass distribution.

We present an algorithm for the deconvolution of isotope-resolved mass spectra of complex peptide mixtures where peaks and isotope series often overlap. The algorithm formulates the problem of mass spectrum deconvolution as a classical statistical problem of variable selection, which aims to interpret the spectrum with the least number of peptides. The LASSO method is used to perform automatic variable selection. The algorithm also makes use of the quantized distribution of peptide masses in the NCBInr database after in silico trypsin digestion as filters to aid the deconvolution process. Errors in the expected isotope pattern are accounted for to avoid spurious isotope series. The effectiveness of the algorithm is demonstrated with annotated ESI spectrum of known peptides for which the peaks and isotope series are highly overlapping. The algorithm successfully finds all correct masses in the experimental spectrum, except for one spectrum where an additional refinement procedure is required to obtain the correct results. Our results compare favorably to those from a widely used commercial program.