Proteomic analysis of limited samples and single cells requires specialized methods that prioritize high sensitivity and minimize sample loss. Consequently, sample preparation is one of the most ...important steps in limited sample analysis workflows to prevent sample loss. In this work, we have eliminated sample handling and transfer steps by processing intact cells directly in the separation capillary, online with capillary electrophoresis coupled to tandem mass spectrometry (CE-MS/MS) for top-down proteomic (TDP) analysis of low numbers of mammalian cancer cells (<10) and single cells. We assessed spray voltage injection of intact cells from a droplet of cell suspension (∼1000 cells) and demonstrated 0-9 intact cells injected with a dependency on the duration of spray voltage application. Spray voltage applied for 2 min injected an average of 7 ± 2 cells and resulted in 33-57 protein and 40-88 proteoform identifications (
= 4). To analyze single cells, manual cell loading by hydrodynamic pressure was used. Replicates of single HeLa cells (
= 4) lysed on the capillary and analyzed by CE-MS/MS demonstrated a range of 17-40 proteins and 23-50 proteoforms identified. An additional cell line, THP-1, was analyzed at the single-cell level, and proteoform abundances were compared to show the capabilities of single-cell TDP (SC-TDP) for assessing cellular heterogeneity. This study demonstrates the initial application of TDP in single-cell proteome-level profiling. These results represent the highest reported identifications from TDP analysis of a single HeLa cell and prove the tremendous potential for CE-MS/MS on-capillary sample processing for high sensitivity analysis of single cells and limited samples.
Tissues and other cell populations are highly heterogeneous at the cellular level, owing to differences in expression and modifications of proteins, polynucleotides, metabolites, and lipids. The ...ability to assess this heterogeneity is crucial in understanding numerous biological phenomena, including various pathologies. Traditional analyses apply bulk-cell sampling, which masks the potentially subtle differences between cells that can be important in understanding of biological processes. These limitations due to cell heterogeneity inspired significant efforts and interest toward the analysis of smaller sample sizes, down to the level of individual cells. Among the emerging techniques, the unique capabilities of capillary electrophoresis coupled with mass spectrometry (CE-MS) made it a prominent technique for proteomics and metabolomics analysis at the single-cell level. In this review, we focus on the application of CE-MS in the proteomic and metabolomic profiling of single cells and highlight the recent advances in sample preparation, separation, MS acquisition, and data analysis.
•Significance of single-cell analysis for elucidating cellular heterogeneity.•CE-MS is an attractive orthogonal technique to current methods for single-cell analysis.•Current alternative single-cell analysis techniques include RPLC, RPLC-MS, direct infusion MS, and MALDI-MS.•Novel applications in single-cell proteomics and metabolomics using CE-MS
In this work, we pioneered the assessment of coupling high-field asymmetric waveform ion mobility spectrometry (FAIMS) with ultrasensitive capillary electrophoresis hyphenated with tandem mass ...spectrometry (CE-MS/MS) to achieve deeper proteome coverage of low nanogram amounts of digested cell lysates. An internal stepping strategy using three or four compensation voltages per analytical run with varied cycle times was tested to determine optimal FAIMS settings and MS parameters for the CE-FAIMS-MS/MS method. The optimized method applied to bottom-up proteomic analysis of 1 ng of HeLa protein digest standard identified 1314 ± 30 proteins, 4829 ± 200 peptide groups, and 7577 ± 163 peptide spectrum matches (PSMs) corresponding to a 16, 25, and 22% increase, respectively, over CE-MS/MS alone, without FAIMS. Furthermore, the percentage of acquired MS/MS spectra that resulted in PSMs increased nearly 2-fold with CE-FAIMS-MS/MS. Label-free quantitation of proteins and peptides was also assessed to determine the precision of replicate analyses from FAIMS methods with increased cycle times. Our results also identified from 1 ng of HeLa protein digest without any prior enrichment 76 ± 9 phosphopeptides, 18% of which were multiphosphorylated. These results represent a 46% increase in phosphopeptide identifications over the control experiments without FAIMS yielding 2.5-fold more multiphosphorylated peptides.
Alarm Safety and Alarm Fatigue Johnson, Kendall R; Hagadorn, James I; Sink, David W
Clinics in perinatology,
09/2017, Volume:
44, Issue:
3
Journal Article
Peer reviewed
Clinical alarm systems have received significant attention in recent years following warnings from hospital accrediting and health care technology organizations regarding patient harm caused by ...unsafe practices. Alarm desensitization or fatigue from frequent, false, or unnecessary alarms, has led to serious events and even patient deaths. Other concerns include settings inappropriate to patient population or condition, inadequate staff training, and improper use or disabling. Research on human factors in alarm response and of functionality of medical devices will help clinicians develop appropriate policies, practices, and device settings for clinical alarms in neonatal intensive care units.
In this work, we developed an ultra-sensitive CE-MS/MS method for bottom-up proteomics analysis of limited samples, down to sub-nanogram levels of total protein. Analysis of 880 and 88 pg of the HeLa ...protein digest standard by CE-MS/MS yielded ∼1100 ± 46 and ∼160 ± 59 proteins, respectively, demonstrating higher protein and peptide identifications than the current state-of-the-art CE-MS/MS-based proteomic analyses with similar amounts of sample. To demonstrate potential applications of our ultra-sensitive CE-MS/MS method for the analysis of limited biological samples, we digested 500 and 1000 HeLa cells using a miniaturized in-solution digestion workflow. From 1-, 5-, and 10-cell equivalents injected from the resulted digests, we identified 744 ± 127, 1139 ± 24, and 1271 ± 6 proteins and 3353 ± 719, 5709 ± 513, and 8527 ± 114 peptide groups, respectively. Furthermore, we performed a comparative assessment of CE-MS/MS and two reversed-phased nano-liquid chromatography (RP-nLC-MS/MS) methods (monolithic and packed columns) for the analysis of a ∼10 ng HeLa protein digest standard. Our results demonstrate complementarity in the protein- and especially peptide-level identifications of the evaluated CE-MS- and RP-nLC-MS-based methods. The techniques were further assessed to detect post-translational modifications and highlight the strengths of the CE-MS/MS approach in identifying potentially important and biologically relevant modified peptides. With a migration window of ∼60 min, CE-MS/MS identified ∼2000 ± 53 proteins on average from a single injection of ∼8.8 ng of the HeLa protein digest standard. Additionally, an average of 232 ± 10 phosphopeptides and 377 ± 14 N-terminal acetylated peptides were identified in CE-MS/MS analyses at this sample amount, corresponding to 2- and 1.5-fold more identifications for each respective modification found by nLC-MS/MS methods.
One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and ...proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.
Phony peach disease (PPD), found predominantly in central and southern Georgia, is a re-emerging disease caused by
(Xf) subsp.
. Accurate detection and rapid removal of symptomatic trees are crucial ...to effective disease management. Currently, peach producers rely solely on visual identification of symptoms to confirm PPD, which can be ambiguous if early in development. We compared visual assessment to quantitative PCR (qPCR) for detecting Xf in 'Julyprince' in 2019 and 2020 (JP2019 and JP2020) and in 'Scarletprince' in 2020 (SP2020). With no prior knowledge of qPCR results, all trees in each orchard were assessed by a cohort of five experienced and five inexperienced raters in the morning and afternoon. Visual identification accuracy of PPD was variable, but experienced raters were more accurate when identifying PPD trees. In JP2019, the mean rater accuracy for experienced and inexperienced raters was 0.882 and 0.805, respectively. For JP2020, the mean rater accuracy for experienced and inexperienced raters was 0.914 and 0.816, respectively. For SP2020, the mean rater accuracy for experienced and inexperienced raters was 0.898 and 0.807, respectively. All raters had false positive (FP) and false negative (FN) observations, but experienced raters had significantly lower FN rates compared with the inexperienced group. Almost all raters overestimated the incidence of PPD in the orchards. Reliability of visual assessments was demonstrated as moderate to good, regardless of experience. Further research is needed to develop accurate and reliable methods of detection to aid management of PPD as both FPs and FNs are costly to peach production.
We examined recent trends and interhospital variation in use of indomethacin, ibuprofen, and surgical ligation for patent ductus arteriosus (PDA) in very-low-birth-weight (VLBW) infants.
Included in ...this retrospective study of the Pediatric Hospital Information System database were 13,853 VLBW infants from 19 US children's hospitals, admitted at age < 3 d between 1 January 2005 and 31 December 2014. PDA management and in-hospital outcomes were examined for trends and variation.
PDA was diagnosed in 5,719 (42%) VLBW infants. Cyclooxygenase inhibitors and/or ligation were used in 74% of infants with PDA overall, however studied hospitals varied greatly in PDA management. Odds of any cyclooxygenase inhibitor or surgical treatment for PDA decreased 11% per year during the study period. This was temporally associated with improved survival but also with increasing bronchopulmonary dysplasia, periventricular leukomalacia, retinopathy of prematurity, and acute renal failure in unadjusted analyses. There was no detectable correlation between hospital-specific changes in PDA management and hospital-specific changes in outcomes of preterm birth during the study period.
Use of cyclooxygenase inhibitors and ligation for PDA in VLBW infants decreased over a 10-y period at the studied hospitals. Further evidence is needed to assess the impact of this change in PDA management.
Peach is an important specialty fruit crop in the United States, and phony peach disease (PPD), caused by
subsp.
, has been a major cause of yield loss since it was first observed in 1885. Under a ...federal eradication program, surveys of PPD were conducted from 1929 to 1972, when the program was terminated. No surveys have been conducted in approximately 50 years; therefore, the current prevalence of PPD in the United States is unknown, especially in the Southeast, where damage was previously most severe. To ascertain the status of PPD, we surveyed orchards in Alabama, Florida, Georgia, and South Carolina from June to August 2020 and, except for South Carolina and northern Georgia, PPD was prevalent. Trees in 17 orchards were subjected to confirmation of
using the AmplifyRP XRT+ for
to corroborate our visual assessments; based on these tests, PPD incidence in the orchards ranged from 0 to 30.5%. Ancillary written surveys of relative PPD presence and prevalence were sent to fruit pathologists from universities in 20 states where PPD was historically reported. Only 35.0% of respondents reported that PPD either currently or recently occurred in their state and, of these, three reported PPD to be of significant concern. The results of the physical and written surveys indicate that PPD remains prevalent mainly in the southeastern region of the United States but, in other states where previously reported, it is either not present or has very low prevalence when compared with historical accounts of the disease.
Abstract
Background
Phony peach disease (PPD) is caused by the plant pathogenic bacterium
Xylella fastidiosa
subsp.
multiplex
(Xfm). Historically, the disease has caused severe yield loss in Georgia ...and elsewhere in the southeastern United States, with millions of PPD trees being removed from peach orchards over the last century. The disease remains a production constraint, and management options are few. Limited research has been conducted on PPD since the 1980s, but the advent of new technologies offers the opportunity for new, foundational research to form a basis for informed management of PPD in the U.S. Furthermore, considering the global threat of Xylella to many plant species, preventing import of Xfm to other regions, particularly where peach is grown, should be considered an important phytosanitary endeavor.
Main topics
We review PPD, its history and impact on peach production, and the eradication efforts that were conducted for 42 years. Additionally, we review the current knowledge of the pathogen, Xfm, and how that knowledge relates to our understanding of the peach—Xylella pathosystem, including the epidemiology of the disease and consideration of the vectors. Methods used to detect the pathogen in peach are discussed, and ramifications of detection in relation to management and control of PPD are considered. Control options for PPD are limited. Our current knowledge of the pathogen diversity and disease epidemiology are described, and based on this, some potential areas for future research are also considered.
Conclusion
There is a lack of recent foundational research on PPD and the associated strain of Xfm. More research is needed to reduce the impact of this pathogen on peach production in the southeastern U.S., and, should it spread internationally, wherever peaches are grown.
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