This study demonstrates how the latest ultrahigh-performance liquid chromatography (UHPLC) technology can be combined with high-resolution accurate-mass (HRAM) mass spectrometry (MS) and long columns packed with fully porous particles to improve bottom-up proteomics analysis with nanoflow liquid chromatography–mass spectrometry (nanoLC-MS) methods. The increased back pressures from the UHPLC system enabled the use of 75 μm I.D. × 75 cm columns packed with 2 μm particles at a typical 300 nL/min flow rate as well as elevated and reduced flow rates. The constant pressure pump operation at 1500 bar reduced sample loading and column washing/equilibration stages and overall overhead time, which maximizes MS utilization time. The versatility of flow rate optimization to balance the sensitivity, throughput with sample loading amount, and capability of using longer gradients contributes to a greater number of peptide and protein identifications for single-shot bottom-up proteomics experiments. The routine proteome profiling and precise quantification of >7000 proteins with single-shot nanoLC-MS analysis open possibilities for large-scale discovery studies with a deep dive into the protein level alterations. Data are available via ProteomeXchange with identifier PXD035665.

Demonstrate the long-term robustness and consistent chromatographic performance of the next-generation Thermo Scientific ™ Vanquish ™ Neo UHPLC System under nanoLC conditions for bottom-up proteome profiling using a 75 µm I.D. × 50 cm Thermo Scientific ™ PepMap ™ Neo Column.

Introduction Peptide analysis with nano-flow gradient separations on long (≥25 cm) analytical columns coupled with HRAM (high-resolution accurate-mass) mass spectrometry is the standard for discoverybased “bottom-up” proteomics research. While delivering unsurpassed sensitivity and depth of analysis, nano-flow setups have yet to achieve the throughput and robustness required for large sample cohort analysis. Therefore, nano-flow LC-MS has been of limited use for translational proteomics, in particular for validation studies.

Demonstrate the superior performance of the Thermo Scientific ™ Vanquish ™ Neo next-generation low-flow UHPLC system for nanoLCMS bottom-up proteome profiling when coupled to a Thermo Scientific ™ Orbitrap Exploris ™ 480 mass spectrometer using the 75 µm I.D. × 75 cm Thermo Scientific ™ EASY-Spray ™ PepMap Neo column. Demonstrate system versatility and potential for new levels of proteomic depth coverage through the coupling of two 75 cm long nano-columns.

Demonstrate the performance of the Thermo Scientific ™ Vanquish ™ Neo UHPLC System, the next−generation nano-, capillary-and micro-flow LC, coupled to a Thermo Scientific ™ Orbitrap Exploris ™ 480 Mass Spectrometer for high-throughput bottom−up proteome profiling using a 75 µm I.D. × 15 cm Thermo Scientific ™ EASY-Spray ™ PepMap ™ Neo Column.

Introduction Nano-flow LC-MS analysis employing long columns and gradients at nano-flow rates has long been established as the method of choice for bottom-up, discovery proteomics. Nevertheless, the challenges associated with nano-flow LC have hitherto limited its application to the field of academic research. The main concerns associated with the technique include locating and eliminating the sources of dead volume, leaks, and capillary or column blockages, combined with the struggle to generate reproducible data. Together, such obstacles can result in the loss of precious time and irreplaceable samples. Another caveat of nanoLC-MS applications is their limited sample throughput and MS utilization. Lower flow rates result in proportionally long periods of MS idle time due to time-consuming sample loading and column equilibration particularly when long separation columns are employed. The latest low-flow UHPLC systems and columns have the potential to overcome many of these challenges and to usher in a new era of robustness and standardization in the field of lowflow LC-MS based analytics.

Introduction Nano-flow LC combined with mass spectrometry has been a mainstay for analyzing proteins and peptides for at least 20 years, particularly for proteomics research. The main advantage that nano-flow brings compared to analytical flow rates is increased sensitivity. Tryptic digestion of biological samples and tissues produces final sample mixtures of low volume, high complexity and with target peptide concentrations ranging from low attomole to high femtomole per microliter levels. In other areas of analytical science, such as clinical research applications, the adoption of nano-flow LC/MS has been limited. In such fields, limited robustness, repeatability, ease-of-use, and throughput are considered the main obstacles to nano-flow LC-MS adoption.1,2 The Vanquish Neo UHPLC system offers a flow range to support applications from nanoto micro-flow, which increases productivity for high sensitivity LC-MS workflows and produces the highest quality results for both LC-MS experts and novice users alike.

Although current LC-MS technology permits scientists to efficiently screen clinical samples in translational research, e.g., steroids, biogenic amines, and even plasma or serum proteomes, in a daily routine, maintaining the balance between throughput and analytical depth is still a limiting factor. A typical approach to enhance the proteome depth is employing offline two-dimensional (2D) fractionation techniques before reversed-phase nanoLC-MS/MS analysis (1D-nanoLC-MS). These additional sample preparation steps usually require extensive sample manipulation, which could result in sample alteration and sample loss. Here, we present and compare 1D-nanoLC-MS with an automated online-2D high-pH RP × low pH RP separation method for deep proteome profiling using a nanoLC system coupled to a high-resolution accurate-mass mass spectrometer. The proof-of-principle study permitted the identification of ca. 500 proteins with ∼10,000 peptides in 15 enzymatically digested crude serum samples collected from healthy donors in 3 laboratories across Europe. The developed method identified 60% more peptides in comparison with conventional 1D nanoLC-MS/MS analysis with ca. 4 times lower throughput while retaining the quantitative information. Serum sample preparation related changes were revealed by applying unsupervised classification techniques and, therefore, must be taken into account while planning multicentric biomarker discovery and validation studies. Overall, this novel method reduces sample complexity and boosts the number of peptide and protein identifications without the need for extra sample handling procedures for samples equivalent to less than 1 μL of blood, which expands the space for potential biomarker discovery by looking deeper into the composition of biofluids.

Mass spectrometry-based (MS) methods are effective tools for discovering protein biomarker candidates that can differentiate between physiological and pathophysiological states. Promising candidates are validated in studies comprising large patient cohorts. Here, targeted protein analytics are used to increase sample throughput. Methods involving antibodies, such as sandwich immunoassays or Western blots, are commonly applied at this stage. Highly-specific and sensitive mass spectrometry-based immunoassays that have been established in recent years offer a suitable alternative to sandwich immunoassays for quantifying proteins. Mass Spectrometric ImmunoAssays (MSIA) and Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA/iMALDI) are two prominent types of MS-based immunoassays in which the capture is done either at the protein or the peptide level. We present an overview of these emerging types of immunoassays and discuss their suitability for the discovery and validation of protein biomarkers. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

The G protein-coupled receptor (GPCR) super-family comprises the largest and most diverse group of membrane receptors in eukaryotes. GPCRs are involved in a plethora of physiological functions in all kinds of tissues. Detailed knowledge about GPCR presence and expression levels in tissues can be very helpful for drug development as the majority of drugs are designed to modulate membrane receptors. Furthermore, it is known that many adverse drug effects result from GPCR interactions. However, very few satisfactory methods are currently available for the detection and quantification of GPCRs. The detection is complicated by their three-dimensional structure, their hydrophobic properties, and their localization in the plasma membrane with 7-trans-membrane domains and small cytosolic and extracellular domains. Due to these properties it is very difficult to generate specific antibodies directed against GPCRs for sandwich immunoassays and Western blot. We therefore designed an immunoaffinity- and mass spectrometry-based approach to analyze GPCR-specific signature peptides in tryptic digests in rat tissue lysates. The expression levels of four different GPCRs were determined using chemically labeled synthetic standard peptides. Here, we demonstrate for the first time, that peptide immunoaffinity MS-based methods can render a reliable and quantitative analysis of multi-membrane spanning receptor molecules.

Blood plasma is a valuable source of potential biomarkers. However, its complexity and the huge dynamic concentration range of its constituents complicate its analysis. To tackle this problem, an immunoprecipitation strategy was employed using antibodies directed against short terminal epitope tags (triple X proteomics antibodies), which allow the enrichment of groups of signature peptides derived from trypsin-digested plasma. Isolated signature peptides are subsequently detected using MALDI-TOF/TOF mass spectrometry. Sensitivity of the immunoaffinity approach was, however, compromised by the presence of contaminant peaks derived from the peptides of nontargeted high abundant proteins. A closer analysis of the enrichment strategy revealed nonspecific peptide binding to the solid phase affinity matrix as the major source of the contaminating peptides. We therefore implemented a sucrose density gradient ultracentrifugation separation step into the procedure. This yielded a 99% depletion of contaminating peptides from a sucrose fraction containing 70% of the peptide-antibody complexes and enabled the detection of the previously undetected low abundance protein filamin-A. Assessment of this novel approach using 15 different triple X proteomics antibodies demonstrated a more consistent detection of a greater number of targeted peptides and a significant reduction in the intensity of nonspecific peptides. Ultracentrifugation coupled with immunoaffinity MS approaches presents a powerful tool for multiplexed plasma protein analysis without the requirement for demanding liquid chromatography separation techniques.

Surfactant deficiency and bronchopulmonary dysplasia (BPD), major obstacles in preterm infants, are addressed with pre‐ and postnatal glucocorticoids which also evoke harmful catabolic side‐effects. Keratinocyte growth factor (KGF) accelerates surfactant production in fetal type II pneumocytes (PN‐II), protects epithelia from injury and is deficient in lungs developing BPD, highlighting its potential efficacy in neonates. Neonatal rats were treated with recombinant human (rh)KGF, betamethasone, or their combination for 48 hr prior to sacrifice after which body weight, surfactant, and tissue phosphatidylcholines (PC) were investigated at postnatal d3, d7, d15, and d21. Pneumocyte proliferation, surfactant protein (SP) expression and SP‐B/C in lung lavage fluid (LLF) were also determined at d7 and d21 to identify broader surfactant changes occurring at the beginning and end of the initial alveolarization phase. While all treatments increased secreted surfactant PC, BM compromised animal growth whereas rhKGF did not. At d3 rhKGF was more effective in male compared to female rats. Single treatments became less effective towards d21. Neither treatment altered PC composition in LLF. BM inhibited PN‐II proliferation and increased surfactant PCs at the expense of tissue PCs. rhKGF however increased surfactant PCs without decreasing other PC species. Whereas SP‐B/C gene expression was induced by all treatments, the changes in secreted SP‐B/C mirrored those observed for surfactant PC. Our results encourage investigation of the mechanisms by which rhKGF improves surfactant homoeostasis, and detailed examination of its efficacy in neonatal lung injury models with a view to implementing it as a non‐catabolic surfactant‐increasing therapeutic in neonatal intensive care. Pediatr. Pulmonol. 2011; 46:883–895. © 2011 Wiley‐Liss, Inc.

Einleitung: Die drei Hauptphosphatidylcholine (PC) des Säuger-Surfactants sind Dipalmitoyl-, Palmitoyl-myristoyl- und Palmitoyl-palmitoleoyl-PC (PC16:0/16:0, PC16:0/14:0 & PC16:0/16:1). Sie bestimmen zusammen mit Surfactantproteinen (SP) die Eigenschaften von Surfactant. Wir adressierten die Regulation & funktionelle Bedeutung von PC16:0/14:0, da es nur im Säuger-Surfactant angereichert wird.

Surfactant predominantly comprises phosphatidylcholine (PC) species, together with phosphatidylglycerols, phosphatidylinositols, neutral lipids, and surfactant proteins-A to -D. Together, dipalmitoyl-PC (PC16:0/16:0), palmitoyl-myristoyl-PC (PC16:0/14:0), and palmitoyl-palmitoleoyl-PC (PC16:0/16:1) make up 75-80% of mammalian surfactant PC, the proportions of which vary during development and in chronic lung diseases. PC16:0/14:0, which exerts specific effects on macrophage differentiation in vitro, increases in surfactant during alveolarization (at the expense of PC16:0/16:0), a prenatal event in humans but postnatal in rats. The mechanisms responsible and the significance of this reversible increase are, however, not understood. We hypothesized that, in rats, myristic acid (C14:0) enriched milk is key to lung-specific PC16:0/14:0 increases in surfactant. We found that surfactant PC16:0/14:0 in suckling rats correlates with C14:0 concentration in plasma chylomicrons and lung tissue triglycerides, and that PC16:0/14:0 fractions reflect exogenous C14:0 supply. Significantly, C14:0 was increased neither in plasma PC, nor in liver triglycerides, free fatty acids, or PC. Lauric acid was also abundant in triglycerides, but was not incorporated into surfactant PC. Comparing a C14:0-rich milk diet with a C14:0-poor carbohydrate diet revealed increased C14:0 and decreased C16:0 in plasma and lung triglycerides, respectively. PC16:0/14:0 enrichment at the expense of PC16:0/16:0 did not impair surfactant surface tension function. However, the PC profile of the alveolar macrophages from the milk-fed animals changed from PC16:0/16:0 rich to PC16:0/14:0 rich. This was accompanied by reduced reactive oxygen species production. We propose that nutritional supply with C14:0 and its lung-specific enrichment may contribute to decreased reactive oxygen species production during alveolarization.

Phosphatidylcholine (PC) synthesis by the direct cytidine diphosphate choline (CDP-choline) pathway in rat liver generates predominantly mono- and di-unsaturated molecular species, while polyunsaturated PC species are synthesized largely by the phosphatidylethanolamine-N-methyltransferase (PEMT) pathway. Although altered PC synthesis has been suggested to contribute to development of hepatocarcinoma and nonalcoholic steatohepatitis, analysis of the specificity of hepatic PC metabolism in human patients has been limited by the lack of sensitive and safe methodologies. Here we incorporated a deuterated methyl-D(9)-labled choline chloride, to quantify biosynthesis fluxes through both of the PC synthetic pathways in vivo in human volunteers and compared these fluxes with those in mice. Rates and molecular specificities of label incorporated into mouse liver and plasma PC were very similar and strongly suggest that label incorporation into human plasma PC can provide a direct measure of hepatic PC synthesis in human subjects. Importantly, we demonstrate for the first time that the PEMT pathway in human liver is selective for polyunsaturated PC species, especially those containing docosahexaenoic acid. Finally, we present a multiple isotopomer distribution analysis approach, based on transfer of deuterated methyl groups to S-adenosylmethionine and subsequent sequential methylations of PE, to quantify absolute flux rates through the PEMT pathway that are applicable to studies of liver dysfunction in clinical studies.

Lung surfactant mainly comprises phosphatidylcholines (PC), together with phosphatidylglycerols and surfactant proteins SP-A to SP-D. Dipalmitoyl-PC (PC16:0/16:0), palmitoylmyristoyl-PC (PC16:0/14:0), and palmitoylpalmitoleoyl-PC (PC16:0/16:1) together comprise 75-80% of surfactant PC. During alveolarization, which occurs postnatally in the rat, PC16:0/14:0 reversibly increases at the expense of PC16:0/16:0. As lipoproteins modify surfactant metabolism, we postulated an extrapulmonary origin of PC16:0/14:0 enrichment in surfactant. We, therefore, fed rats (d19-26) with trilaurin (C12:0(3)), trimyristin (C14:0(3)), tripalmitin (C16:0(3)), triolein (C18:1(3)) or trilinolein (C18:2(3)) vs. carbohydrate diet to assess their effects on surfactant PC composition and surface tension function using a captive bubble surfactometer. Metabolism was assessed with deuterated C12:0 (ω-d(3)-C12:0) and ω-d(3)-C14:0. C14:0(3) increased PC16:0/14:0 in surfactant from 12 ± 1 to 45 ± 3% and decreased PC16:0/16:0 from 47 ± 1 to 29 ± 2%, with no impairment of surface tension function. Combined phospholipase A(2) assay and mass spectrometry revealed that 50% of the PC16:0/14:0 peak comprised its isomer 1-myristoyl-2-palmitoyl-PC (PC14:0/16:0). While C12:0(3) was excluded from incorporation into PC, it increased PC16:0/14:0 as well. C16:0(3), C18:1(3), and C18:2(3) had no significant effect on PC16:0/16:0 or PC16:0/14:0. d(3)-C14:0 was enriched in lung PC, either via direct supply or via d(3)-C12:0 elongation. Enrichment of d(3)-C14:0 in surfactant PC contrasted its rapid turnover in plasma and liver PC, where its elongation product d(3)-C16:0 surmounted d(3)-C14:0. In summary, high surfactant PC16:0/14:0 during lung development correlates with C14:0 and C12:0 supply via specific C14:0 enrichment into lung PC. Surfactant that is high in PC16:0/14:0 but low in PC16:0/16:0 is compatible with normal respiration and surfactant function in vitro.

Pediatric PulmonologyVolume 44, Issue 12 p. 1157-1158 Invited Commentary Therapeutic lung surfactants as carriers for other therapeutics—a matter of vision, courage and determination Wolfgang Bernhard MD, PhD, Corresponding Author Wolfgang Bernhard MD, PhD wolfgang.bernhard@med.uni-tuebingen.de Department of Neonatology, Faculty of Medicine, Eberhard-Karls-University, Calwer Straße 7 & Otfried-Müller-Str. 27, D-72076 Tübingen, GermanyDepartment of Neonatology, Faculty of Medicine, Eberhard-Karls-University, Calwer Straße 7 & Otfried-Müller-Str. 27, D-72076 Tübingen, Germany.Search for more papers by this authorChristopher J. Pynn PhD, Christopher J. Pynn PhD Department of Neonatology, Faculty of Medicine, Eberhard-Karls-University, Calwer Straße 7 & Otfried-Müller-Str. 27, D-72076 Tübingen, GermanySearch for more papers by this author Wolfgang Bernhard MD, PhD, Corresponding Author Wolfgang Bernhard MD, PhD wolfgang.bernhard@med.uni-tuebingen.de Department of Neonatology, Faculty of Medicine, Eberhard-Karls-University, Calwer Straße 7 & Otfried-Müller-Str. 27, D-72076 Tübingen, GermanyDepartment of Neonatology, Faculty of Medicine, Eberhard-Karls-University, Calwer Straße 7 & Otfried-Müller-Str. 27, D-72076 Tübingen, Germany.Search for more papers by this authorChristopher J. Pynn PhD, Christopher J. Pynn PhD Department of Neonatology, Faculty of Medicine, Eberhard-Karls-University, Calwer Straße 7 & Otfried-Müller-Str. 27, D-72076 Tübingen, GermanySearch for more papers by this author First published: 12 November 2009 https://doi.org/10.1002/ppul.21123Citations: 2AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume44, Issue12December 2009Pages 1157-1158 RelatedInformation