Cellular and molecular architecture of submucosal glands in wild-type and cystic fibrosis pigs.

Submucosal glands (SMGs) protect lungs but can also contribute to disease. For example, in cystic fibrosis (CF), SMGs produce abnormal mucus that disrupts mucociliary transport. CF is an ion transport disease, yet knowledge of the ion transporters expressed by SMG acini, which produce mucus, and SMG ducts that carry it to the airway lumen is limited. Therefore, we isolated SMGs from newborn pigs and used single-cell messenger RNA sequencing, immunohistochemistry, and in situ hybridization to identify cell types, gene expression, and spatial distribution. Cell types and transcript levels were the same in non-CF and CF SMGs, suggesting that loss of epithelial anion secretion rather than an intrinsic cell defect causes CF mucus abnormalities. Gene signatures of acinar mucous and acinar serous cells revealed specialized functions in producing mucins and antimicrobials, respectively. However, surprisingly, these two cell types expressed the same ion transporters and neurohumoral receptors, suggesting the importance of balancing mucin and liquid secretion to produce optimal mucus properties. SMG duct cell transcripts suggest that they secrete HCO3- and Cl-, and thus have some similarity to pancreatic ducts that are also defective in CF. These and additional findings suggest the functions of the SMG acinus and duct and provide a baseline for understanding how environmental and genetic challenges impact their contribution to lung disease.

Single-molecule long-read sequencing reveals the chromatin basis of gene expression.

Genome-wide chromatin accessibility and nucleosome occupancy profiles have been widely investigated, while the long-range dynamics remain poorly studied at the single-cell level. Here, we present a new experimental approach, methyltransferase treatment followed by single-molecule long-read sequencing (MeSMLR-seq), for long-range mapping of nucleosomes and chromatin accessibility at single DNA molecules and thus achieve comprehensive-coverage characterization of the corresponding heterogeneity. MeSMLR-seq offers direct measurements of both nucleosome-occupied and nucleosome-evicted regions on a single DNA molecule, which is challenging for many existing methods. We applied MeSMLR-seq to haploid yeast, where single DNA molecules represent single cells, and thus we could investigate the combinatorics of many (up to 356) nucleosomes at long range in single cells. We illustrated the differential organization principles of nucleosomes surrounding the transcription start site for silent and actively transcribed genes, at the single-cell level and in the long-range scale. The heterogeneous patterns of chromatin status spanning multiple genes were phased. Together with single-cell RNA-seq data, we quantitatively revealed how chromatin accessibility correlated with gene transcription positively in a highly heterogeneous scenario. Moreover, we quantified the openness of promoters and investigated the coupled chromatin changes of adjacent genes at single DNA molecules during transcription reprogramming. In addition, we revealed the coupled changes of chromatin accessibility for two neighboring glucose transporter genes in response to changes in glucose concentration.

Early pathogenesis of cystic fibrosis gallbladder disease in a porcine model.

Hepatobiliary disease causes significant morbidity in people with cystic fibrosis (CF), yet this problem remains understudied. We previously found that newborn CF pigs have microgallbladders with significant luminal obstruction in the absence of infection and consistent inflammation. In this study, we sought to better understand the early pathogenesis of CF pig gallbladder disease. We hypothesized that loss of CFTR would impair gallbladder epithelium anion/liquid secretion and increase mucin production. CFTR was expressed apically in non-CF pig gallbladder epithelium but was absent in CF. CF pig gallbladders lacked cAMP-stimulated anion transport. Using a novel gallbladder epithelial organoid model, we found that Cl- or HCO3- was sufficient for non-CF organoid swelling. This response was absent for non-CF organoids in Cl-/HCO3--free conditions and in CF. Single-cell RNA-sequencing revealed a single epithelial cell type in non-CF gallbladders that coexpressed CFTR, MUC5AC, and MUC5B. Despite CF gallbladders having increased luminal MUC5AC and MUC5B accumulation, there was no significant difference in the epithelial expression of gel-forming mucins between non-CF and CF pig gallbladders. In conclusion, these data suggest that loss of CFTR-mediated anion transport and fluid secretion contribute to microgallbladder development and luminal mucus accumulation in CF.

Widespread airway distribution and short-term phenotypic correction of cystic fibrosis pigs following aerosol delivery of piggyBac/adenovirus.

Cystic fibrosis (CF) is a common genetic disease caused by mutations in the gene coding for cystic fibrosis transmembrane conductance regulator (CFTR). Although CF affects multiple organ systems, chronic bacterial infections and inflammation in the lung are the leading causes of morbidity and mortality in people with CF. Complementation with a functional CFTR gene repairs this defect, regardless of the disease-causing mutation. In this study, we used a gene delivery system termed piggyBac/adenovirus (Ad), which combines the delivery efficiency of an adenoviral-based vector with the persistent expression of a DNA transposon-based vector. We aerosolized piggyBac/Ad to the airways of pigs and observed widespread pulmonary distribution of vector. We quantified the regional distribution in the airways and observed transduction of large and small airway epithelial cells of non-CF pigs, with ∼30-50% of surface epithelial cells positive for GFP. We transduced multiple cell types including ciliated, non-ciliated, basal, and submucosal gland cells. In addition, we phenotypically corrected CF pigs following delivery of piggyBac/Ad expressing CFTR as measured by anion channel activity, airway surface liquid pH, and bacterial killing ability. Combining an integrating DNA transposon with adenoviral vector delivery is an efficient method for achieving functional CFTR correction from a single vector administration.

Gel-forming mucins form distinct morphologic structures in airways.

Gel-forming mucins, the primary macromolecular components of airway mucus, facilitate airway clearance by mucociliary transport. In cystic fibrosis (CF) altered mucus properties impair mucociliary transport. Airways primarily secrete two closely related gel-forming mucins, MUC5B and MUC5AC. However, their morphologic structures and associations in airways that contain abundant submucosal glands and goblet cells are uncertain. Moreover, there is limited knowledge about mucins in airways not affected by inflammation, infection, or remodeling or in CF airways. Therefore, we examined airways freshly excised from newborn non-CF pigs and CF pigs before secondary manifestations develop. We found that porcine submucosal glands produce MUC5B, whereas goblet cells produce predominantly MUC5AC plus some MUC5B. We found that MUC5B emerged from submucosal gland ducts in the form of strands composed of multiple MUC5B filaments. In contrast, MUC5AC emerged from goblet cells as wispy threads and sometimes formed mucin sheets. In addition, MUC5AC often partially coated the MUC5B strands. Compared with non-CF, MUC5B more often filled CF submucosal gland ducts. MUC5AC sheets also accumulated in CF airways overlying MUC5B strands. These results reveal distinct morphology and interactions for MUC5B and MUC5AC and suggest that the two mucins make distinct contributions to mucociliary transport. Thus, they provide a framework for understanding abnormalities in disease.

Interferon-γ Inhibits Ebola Virus Infection.

Ebola virus outbreaks, such as the 2014 Makona epidemic in West Africa, are episodic and deadly. Filovirus antivirals are currently not clinically available. Our findings suggest interferon gamma, an FDA-approved drug, may serve as a novel and effective prophylactic or treatment option. Using mouse-adapted Ebola virus, we found that murine interferon gamma administered 24 hours before or after infection robustly protects lethally-challenged mice and reduces morbidity and serum viral titers. Furthermore, we demonstrated that interferon gamma profoundly inhibits Ebola virus infection of macrophages, an early cellular target of infection. As early as six hours following in vitro infection, Ebola virus RNA levels in interferon gamma-treated macrophages were lower than in infected, untreated cells. Addition of the protein synthesis inhibitor, cycloheximide, to interferon gamma-treated macrophages did not further reduce viral RNA levels, suggesting that interferon gamma blocks life cycle events that require protein synthesis such as virus replication. Microarray studies with interferon gamma-treated human macrophages identified more than 160 interferon-stimulated genes. Ectopic expression of a select group of these genes inhibited Ebola virus infection. These studies provide new potential avenues for antiviral targeting as these genes that have not previously appreciated to inhibit negative strand RNA viruses and specifically Ebola virus infection. As treatment of interferon gamma robustly protects mice from lethal Ebola virus infection, we propose that interferon gamma should be further evaluated for its efficacy as a prophylactic and/or therapeutic strategy against filoviruses. Use of this FDA-approved drug could rapidly be deployed during future outbreaks.

Characterization of fusion genes and the significantly expressed fusion isoforms in breast cancer by hybrid sequencing.

We developed an innovative hybrid sequencing approach, IDP-fusion, to detect fusion genes, determine fusion sites and identify and quantify fusion isoforms. IDP-fusion is the first method to study gene fusion events by integrating Third Generation Sequencing long reads and Second Generation Sequencing short reads. We applied IDP-fusion to PacBio data and Illumina data from the MCF-7 breast cancer cells. Compared with the existing tools, IDP-fusion detects fusion genes at higher precision and a very low false positive rate. The results show that IDP-fusion will be useful for unraveling the complexity of multiple fusion splices and fusion isoforms within tumorigenesis-relevant fusion genes.

Epigenetic silencing of the human NOS2 gene: rethinking the role of nitric oxide in human macrophage inflammatory responses.

Macrophages, including alveolar macrophages, are primary phagocytic cells of the innate immune system. Many studies of macrophages and inflammation have been done in mouse models, in which inducible NO synthase (NOS2) and NO are important components of the inflammatory response. Human macrophages, in contrast to mouse macrophages, express little detectable NOS2 and generate little NO in response to potent inflammatory stimuli. The human NOS2 gene is highly methylated around the NOS2 transcription start site. In contrast, mouse macrophages contain unmethylated cytosine-phosphate-guanine (CpG) dinucleotides proximal to the NOS2 transcription start site. Further analysis of chromatin accessibility and histone modifications demonstrated a closed conformation at the human NOS2 locus and an open conformation at the murine NOS2 locus. In examining the potential for CpG demethylation at the NOS2 locus, we found that the human NOS2 gene was resistant to the effects of demethylation agents both in vitro and in vivo. Our data demonstrate that epigenetic modifications in human macrophages are associated with CpG methylation, chromatin compaction, and histone modifications that effectively silence the NOS2 gene. Taken together, our findings suggest there are significant and underappreciated differences in how murine and human macrophages respond to inflammatory stimuli.

A microRNA processing defect in smokers' macrophages is linked to SUMOylation of the endonuclease DICER.

Despite the fact that alveolar macrophages play an important role in smoking-related disease, little is known about what regulates their pathophysiologic phenotype. Evaluating smoker macrophages, we found significant down-regulation of multiple microRNAs (miRNAs). This work investigates the hypothesis that cigarette smoke alters mature miRNA expression in lung macrophages by inhibiting processing of primary miRNA transcripts. Studies on smoker alveolar macrophages showed a defect in miRNA maturation. Studies on the miRNA biogenesis machinery led us to focus on the cytosolic RNA endonuclease, DICER. DICER cleaves the stem-loop structure from pre-miRNAs, allowing them to dissociate into their mature 20-22-nucleotide single-stranded form. DICER activity assays confirmed impaired DICER activity following cigarette smoke exposure. Further protein studies demonstrated a decreased expression of the native 217-kDa form of DICER and an accumulation of high molecular weight forms with cigarette smoke exposure. This molecular mass shift was shown to contain SUMO moieties and could be blocked by silencing RNA directed at the primary SUMOylating ligase, Ubc9. In determining the cigarette smoke components responsible for changes in DICER, we found that N-acetylcysteine, an antioxidant and anti-aldehyde, protected DICER protein and activity from cigarette smoke extract. This massive down-regulation of miRNAs (driven in part by alterations in DICER) may be an important regulator of the disease-promoting macrophage phenotype found in the lungs of smokers.

Inositol-requiring enzyme 1 inhibits respiratory syncytial virus replication.

Despite being a major health problem, respiratory syncytial virus (RSV) infections remain without specific therapy. Identification of novel host cellular responses that play a role in the pathogenesis of RSV infection is needed for therapeutic development. The endoplasmic reticulum (ER) stress response is an evolutionarily conserved cellular signaling cascade that has been implicated in multiple biological phenomena, including the pathogenesis of some viral infections. In this study, we investigate the role of the ER stress response in RSV infection using an in vitro A549 cell culture model. We found that RSV infection induces a non-canonical ER stress response with preferential activation of the inositol-requiring enzyme 1 (IRE1) and activated transcription factor 6 (ATF6) pathways with no concomitant significant activation of the protein kinase R-like ER kinase (PERK) pathway. Furthermore, we discovered that IRE1 has an inhibitory effect on RSV replication. Our data characterize, for the first time, the nature of the ER stress response in the setting of RSV infection and identify the IRE1 stress pathway as a novel cellular anti-RSV defense mechanism.

Effects of prenatal inhalation exposure to copper nanoparticles on murine dams and offspring.

BACKGROUND: Increasing numbers of individuals may be exposed to nanomaterials during pregnancy. The overarching goal of this investigation was to determine if prenatal inhalation exposure to copper nanoparticles (Cu NPs) has an effect on dams and offspring, including an analysis of inflammatory markers (Th1/Th2 cytokine profiles). METHODS: Physicochemical characterization of Cu NPs was performed. Pregnant and non-pregnant mice (C57Bl/6 J) were exposed to Cu NPs or laboratory air in the whole-body chamber for 4 hrs/day on gestation days (GD) 3-19 (3.5 mg/m(3)). Animals were euthanized on GD 19 (0 week) or 7 weeks later. Bronchoalveolar lavage (BAL) fluid was analyzed for total and differential cells. Cytokine/chemokine concentrations were determined in the BAL fluid and the plasma of dams/non-pregnant mice and pups. Cu content was determined in the lungs and the blood of dams/non-pregnant mice and pups, in the placentas as well as in the whole bodies of pups immediately after delivery. Lungs and placentas were evaluated for histopathological changes. Gene expression of the Th1/Th2 profiles were analyzed in spleens of pups. RESULTS: The survival rate of 7 week old pups exposed to Cu NPs was significantly lower than control pups (73 vs. 97 %). The average litter size, male/female ratio, body weight and lenght at birth were not different between Cu NP-exposed and control mice. Both pregnant and non-pregnant mice exposed to Cu NPs had significant pulmonary inflammation with increased number of neutrophils in the BAL fluid compared to controls. Perivascular lymphoplasmacytic cuffing was found in the lungs of exposed mice and was more pronounced in the non-pregnant group. Similarly, levels of inflammatory cytokines/chemokines IL-12(p40), G-CSF, GM-CSF, KC, MCP-1, MIP-1α, MIP-1ß, RANTES and TNF-α in BAL fluid were significantly higher in non-pregnant than pregnant exposed mice. Histopathology evaluation of placentas did not identify any pathological changes. No translocation of Cu into the placenta or the fetus was found by inductively coupled plasma-mass spectroscopy. Expression of several Th1/Th2 or other immune response genes in pups' spleens were found to be significantly up- or down-regulated. CONCLUSIONS: Prenatal exposure to Cu NPs caused a profound pulmonary inflammation in dams and strong immunomodulatory effects in offspring. There was no clear polarization of genes expressed in pups' spleens towards Th1 or Th2 type of response.

Pancreatic enzymes digest obstructive meconium from cystic fibrosis pig intestines.

Introduction: Meconium ileus (MI) is a life-threatening obstruction of the intestines affecting ∼15% of newborns with cystic fibrosis (CF). Current medical treatments for MI often fail, requiring surgical intervention. MI typically occurs in newborns with pancreatic insufficiency from CF. Meconium contains mucin glycoprotein, a potential substrate for pancreatic enzymes or mucolytics. Our study aim was to determine whether pancreatic enzymes in combination with mucolytic treatments dissolve obstructive meconium using the CF pig model. Methods: We collected meconium from CF pigs at birth and submerged it in solutions with and without pancreatic enzymes, including normal saline, 7% hypertonic saline, and the reducing agents N-acetylcysteine (NAC) and dithiothreitol (DTT). We digested meconium at 37 °C with agitation, and measured meconium pigment release by spectrophotometry and residual meconium solids by filtration. Results and discussion: In CF pigs, meconium appeared as a solid pigmented mass obstructing the ileum. Meconium microscopically contained mucus glycoprotein, cellular debris, and bile pigments. Meconium fragments released pigments with maximal absorption at 405 nm after submersion in saline over approximately 8 h. Pancreatic enzymes significantly increased pigment release and decreased residual meconium solids. DTT did not improve meconium digestion and the acidic reducing agent NAC worsened digestion. Pancreatic enzymes digested CF meconium best at neutral pH in isotonic saline. We conclude that pancreatic enzymes digest obstructive meconium from CF pigs, while hydrating or reducing agents alone were less effective. This work suggests a potential role for pancreatic enzymes in relieving obstruction due to MI in newborns with CF.

Arteriovenous metabolomics in pigs reveals CFTR regulation of metabolism in multiple organs.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), a multiorgan disease that is characterized by diverse metabolic defects. However, other than specific CFTR mutations, the factors that influence disease progression and severity remain poorly understood. Aberrant metabolite levels have been reported, but whether CFTR loss itself or secondary abnormalities (infection, inflammation, malnutrition, and various treatments) drive metabolic defects is uncertain. Here, we implemented comprehensive arteriovenous metabolomics in newborn CF pigs, and the results revealed CFTR as a bona fide regulator of metabolism. CFTR loss impaired metabolite exchange across organs, including disruption of lung uptake of fatty acids, yet enhancement of uptake of arachidonic acid, a precursor of proinflammatory cytokines. CFTR loss also impaired kidney reabsorption of amino acids and lactate and abolished renal glucose homeostasis. These and additional unexpected metabolic defects prior to disease manifestations reveal a fundamental role for CFTR in controlling multiorgan metabolism. Such discovery informs a basic understanding of CF, provides a foundation for future investigation, and has implications for developing therapies targeting only a single tissue.

Influenza A viral replication is blocked by inhibition of the inositol-requiring enzyme 1 (IRE1) stress pathway.

Known therapies for influenza A virus infection are complicated by the frequent emergence of resistance. A therapeutic strategy that may escape viral resistance is targeting host cellular mechanisms involved in viral replication and pathogenesis. The endoplasmic reticulum (ER) stress response, also known as the unfolded protein response (UPR), is a primitive, evolutionary conserved molecular signaling cascade that has been implicated in multiple biological phenomena including innate immunity and the pathogenesis of certain viral infections. We investigated the effect of influenza A viral infection on ER stress pathways in lung epithelial cells. Influenza A virus induced ER stress in a pathway-specific manner. We showed that the virus activates the IRE1 pathway with little or no concomitant activation of the PERK and the ATF6 pathways. When we examined the effects of modulating the ER stress response on the virus, we found that the molecular chaperone tauroursodeoxycholic acid (TUDCA) significantly inhibits influenza A viral replication. In addition, a specific inhibitor of the IRE1 pathway also blocked viral replication. Our findings constitute the first evidence that ER stress plays a role in the pathogenesis of influenza A viral infection. Decreasing viral replication by modulating the host ER stress response is a novel strategy that has important therapeutic implications.

Identification of an autophagy defect in smokers' alveolar macrophages.

Alveolar macrophages are essential for clearing bacteria from the alveolar surface and preventing microbe-induced infections. It is well documented that smokers have an increased incidence of infections, in particular lung infections. Alveolar macrophages accumulate in smokers' lungs, but they have a functional immune deficit. In this study, we identify an autophagy defect in smokers' alveolar macrophages. Smokers' alveolar macrophages accumulate both autophagosomes and p62, a marker of autophagic flux. The decrease in the process of autophagy leads to impaired protein aggregate clearance, dysfunctional mitochondria, and defective delivery of bacteria to lysosomes. This study identifies the autophagy pathway as a potential target for interventions designed to decrease infection rates in smokers and possibly in individuals with high environmental particulate exposure.

Obstructive sleep apnea predicts 10-year cardiovascular disease-related mortality in the Sleep Heart Health Study: a machine learning approach.

STUDY OBJECTIVES: Obstructive sleep apnea (OSA) is considered to be an important risk factor for the development of cardiovascular disease (CVD). This study aimed to develop and evaluate a machine learning approach with a set of features for assessing the 10-year CVD mortality risk of the OSA population. METHODS: This study included 2,464 patients with OSA who met study inclusion criteria and were selected from the Sleep Heart Health Study. We evaluated the importance of potential features by mutual information. The top 9 features were selected to develop a random forest model. RESULTS: We evaluated the model performance on a test set (n = 493) using the area under the receiver operating curve with 95% confidence interval and confusion matrix. A random forest model awarded the highest area under the receiver operating curve of 0.84 (95% confidence interval: 0.78-0.89). The specificity and sensitivity were 73.94% and 81.82%, respectively. Sixty-three years old was a threshold for increased risk of 10-year CVD mortality. Persons with severe OSA had higher risk than those with mild OSA. CONCLUSIONS: This study demonstrated that a random forest model can provide a quick assessment of the risk of 10-year CVD mortality. Our model may be more informative for patients with OSA in determining their future CVD mortality risk. CITATION: Li A, Roveda JM, Powers LS, Quan SF. Obstructive sleep apnea predicts 10-year cardiovascular disease-related mortality in the Sleep Heart Health Study: a machine learning approach. J Clin Sleep Med. 2022;18(2):497-504.

A deep learning-based algorithm for detection of cortical arousal during sleep.

STUDY OBJECTIVES: The frequency of cortical arousals is an indicator of sleep quality. Additionally, cortical arousals are used to identify hypopneic events. However, it is inconvenient to record electroencephalogram (EEG) data during home sleep testing. Fortunately, most cortical arousal events are associated with autonomic nervous system activity that could be observed on an electrocardiography (ECG) signal. ECG data have lower noise and are easier to record at home than EEG. In this study, we developed a deep learning-based cortical arousal detection algorithm that uses a single-lead ECG to detect arousal during sleep. METHODS: This study included 1,547 polysomnography records that met study inclusion criteria and were selected from the Multi-Ethnic Study of Atherosclerosis database. We developed an end-to-end deep learning model consisting of convolutional neural networks and recurrent neural networks which: (1) accepted varying length physiological data; (2) directly extracted features from the raw ECG signal; (3) captured long-range dependencies in the physiological data; and (4) produced arousal probability in 1-s resolution. RESULTS: We evaluated the model on a test set (n = 311). The model achieved a gross area under precision-recall curve score of 0.62 and a gross area under receiver operating characteristic curve score of 0.93. CONCLUSION: This study demonstrated the end-to-end deep learning approach with a single-lead ECG has the potential to be used to accurately detect arousals in home sleep tests.

Sleep patterns and sleep deprivation recorded by actigraphy in 4th-grade and 5th-grade students.

OBJECTIVE: This study investigates sleep patterns of fourth- and fifth-grade students using actigraphy. METHODS: The study included 257 students enrolled in a Southwestern US school district who participated in a novel sleep science curriculum during the Spring 2016-17 and Fall 2017-18 semesters and met the study inclusion criteria. As part of this curriculum, participants underwent 5-7 days of continuous wrist actigraphy and completed an online sleep diary. RESULTS: Approximately two-thirds of the 9-11-year-old fourth- and fifth-grade students slept less than the minimum 9 h per night recommended by both the American Academy of Sleep Medicine/Sleep Research Society and the National Sleep Foundation. The sleep midpoint time on weekends was about 1 h later than on weekdays. There was a significant effect of age on sleep duration. Compared to 9-year old students, a larger proportion of 10-year old students had a sleep duration less than 8.5 h. Boys had shorter sleep duration than girls, and a larger percentage of boys obtained less than 9 h of sleep compared to girls. CONCLUSIONS: Insufficient sleep is a highly prevalent condition among 9-11-year-old fourth- and fifth-grade elementary students. Importantly, there is a difference between sleep patterns on weekdays and weekends which may portend greater problems with sleep in adolescence and young adulthood.

Lack of airway submucosal glands impairs respiratory host defenses.

Submucosal glands (SMGs) are a prominent structure that lines human cartilaginous airways. Although it has been assumed that SMGs contribute to respiratory defense, that hypothesis has gone without a direct test. Therefore, we studied pigs, which have lungs like humans, and disrupted the gene for ectodysplasin (EDA-KO), which initiates SMG development. EDA-KO pigs lacked SMGs throughout the airways. Their airway surface liquid had a reduced ability to kill bacteria, consistent with SMG production of antimicrobials. In wild-type pigs, SMGs secrete mucus that emerges onto the airway surface as strands. Lack of SMGs and mucus strands disrupted mucociliary transport in EDA-KO pigs. Consequently, EDA-KO pigs failed to eradicate a bacterial challenge in lung regions normally populated by SMGs. These in vivo and ex vivo results indicate that SMGs are required for normal antimicrobial activity and mucociliary transport, two key host defenses that protect the lung.

Affinity capture mass spectrometry of biomarker proteins using peptide ligands from biopanning.

Affinity capture mass spectrometry was used to isolate and ionize protein A from Staphylococcus aureus from both a commercial source and cell culture lysate using matrix assisted laser desorption/ionization (MALDI) mass spectrometry. Two surfaces are compared: gold surfaces with immunoglobulin G covalently immobilized and silica surfaces with a covalently bound small peptide discovered via biopanning. A detection limit of 2.22 bacterial cells/mL of culture fluid was determined for the immobilized peptide surfaces. This study emphasizes the ability to use peptide ligands to effectively capture a biomarker protein out of a complex mixture. This demonstrates the potential to use biopanning to generate capture ligands for a large variety of target proteins and subsequently detect the captured protein using MALDI mass spectrometry.