Microflow Liquid Chromatography Research Articles

High-Throughput Proteomics and Phosphoproteomics of Rat Tissues Using Microflow Zeno SWATH.

High-throughput tissue proteomics has great potential in the advancement of precision medicine. Here, we investigated the combined sensitivity of trap-elute microflow liquid chromatography with a ZenoTOF for DIA proteomics and phosphoproteomics. Method optimization was conducted on HEK293T cell lines to determine the optimal variable window size, MS2 accumulation time and gradient length. The ZenoTOF 7600 was then compared to the previous generation TripleTOF 6600 using eight rat organs, finding up to 23% more proteins using a fifth of the sample load and a third of the instrument time. Spectral reference libraries generated from Zeno SWATH data in FragPipe (MSFragger-DIA/DIA-NN) contained 4 times more fragment ions than the DIA-NN only library and quantified more proteins. Replicate single-shot phosphopeptide enrichments of 50-100 μg of rat tryptic peptide were analyzed by microflow HPLC using Zeno SWATH without fractionation. Using Spectronaut we quantified a shallow phosphoproteome containing 1000-3000 phosphoprecursors per organ. Promisingly, clear hierarchical clustering of organs was observed with high Pearson correlation coefficients >0.95 between replicate enrichments and median CV of 20%. The combined sensitivity of microflow HPLC with Zeno SWATH allows for the high-throughput quantitation of an extensive proteome and shallow phosphoproteome from small tissue samples.

High-Throughput Microbore LC-MS Lipidomics to Investigate APOE Phenotypes.

Microflow liquid chromatography interfaced with mass spectrometry (μLC-MS/MS) is increasingly applied for high-throughput profiling of biological samples and has been proven to have an acceptable trade-off between sensitivity and reproducibility. However, lipidomics applications are scarce. We optimized a μLC-MS/MS system utilizing a 1 mm inner diameter × 100 mm column coupled to a triple quadrupole mass spectrometer to establish a sensitive, high-throughput, and robust single-shot lipidomics workflow. Compared to conventional lipidomics methods, we achieve a ∼4-fold increase in response, facilitating quantification of 351 lipid species from a single iPSC-derived cerebral organoid during a 15 min LC-MS analysis. Consecutively, we injected 303 samples over ∼75 h to prove the robustness and reproducibility of the microflow separation. As a proof of concept, μLC-MS/MS analysis of Alzheimer's disease patient-derived iPSC cerebral organoid reveals differential lipid metabolism depending on APOE phenotype (E3/3 vs E4/4). Microflow separation proves to be an environmentally friendly and cost-effective method as it reduces the consumption of harmful solvents. Also, the data demonstrate robust, in-depth, high-throughput performance to enable routine clinical or biomedical applications.

Comprehensive species- and processing-specific peptide profiling of pasteurized, extended shelf-life and ultra-high temperature milk from cow, goat, sheep, buffalo, and mare

The present study aimed to identify endogenous milk peptides for species differentiation independent of heat exposure. Thus, comprehensive milk peptide profiles from five species and three types of heat treatments were analyzed by micro-flow liquid chromatography ion mobility quadrupole time-of-flight mass spectrometry (microLC-IM-QTOF) with subsequent database search leading to ≥ 3000 identified peptides. In the milks, 1154 peptides were unique for cow, 712 for sheep, 466 for goat, 197 for buffalo, and 69 for mare. Most peptides were detected in extended-shelf life (ESL) milk (2010), followed by ultra-high temperature (UHT) processed (1474) and pasteurized milk (1459 peptides), with 693 peptides present in all milk types. A blind test set of 64 samples confirmed eight species-specific, but heat-independent marker peptides in milk from cow, seven from goat, six from sheep, nine from buffalo, and three from mare. The generated peptide profiles can also be used to identify species- and heat-specific markers.

Abstract 18472: Plasma Proteome Changes in Heart Failure and Non-Heart Failure Patients Undergoing Cardiac Surgery

Introduction: Patients with and without heart failure display different clinical courses after cardiac surgery. Hypothesis: Unbiased analyses of the plasma proteome from extreme phenotypes in myocardial function may identify differences in candidate proteins differentially modified by cardiac surgery using cardiopulmonary bypass (CPB). Methods: We combined two prospectively-collected datasets: Left ventricular assist device (LVAD) recipients from the INSPIRE-FLO trial (N=94, NCT03081052) and coronary artery bypass graft (CABG) surgery patients with normal biventricular function (N=50). For each population, we obtained EDTA-plasma from arterial blood sampling in the operating room and on postoperative day 1. Including quality control pools, 304 total samples were digested in a 96-well format with trypsin. Peptides were analyzed by microflow liquid chromatography coupled to tandem mass spectrometry using data-independent acquisition. Proteins were identified and quantified at a 1% false discovery rate (FDR) in Spectronaut. Statistical analyses utilized a t-test with FDR correction (significance defined by log2FC>1 and q<0.05). Results: There were 943 protein groups quantified in at least one plasma sample. Patients undergoing LVAD surgery had significantly higher levels of 135 proteins including acute phase reactants (CRP, SAA1, SAA2), putative neutrophil-derived proteins (S100A8/9) and markers of renal insufficiency (B2M and REG3A) (Figure 1). Cardiac surgery using cardiopulmonary bypass increased acute phase and inflammatory markers, and cardiac proteins (ENOB, MYG and KCRM), with NGAL, TIMP1 and PAI1 among proteins with higher absolute increase after LVAD implantation versus CABG surgery ( Fig.1 ). Conclusions: Unbiased analyses of the plasma proteome identifies baseline and postoperative differences associated with extreme phenotypes of myocardial function undergoing cardiac surgery using CPB.

A region-resolved proteomic map of the human brain enabled by high-throughput proteomics.

Substantial efforts are underway to deepen our understanding of human brain morphology, structure, and function using high-resolution imaging as well as high-content molecular profiling technologies. The current work adds to these approaches by providing a comprehensive and quantitative protein expression map of 13 anatomically distinct brain regions covering more than 11,000 proteins. This was enabled by the optimization, characterization, and implementation of a high-sensitivity and high-throughput microflow liquid chromatography timsTOF tandem mass spectrometry system (LC-MS/MS) capable of analyzing more than 2,000 consecutive samples prepared from formalin-fixed paraffin embedded (FFPE) material. Analysis of this proteomic resource highlighted brain region-enriched protein expression patterns and functional protein classes, protein localization differences between brain regions and individual markers for specific areas. To facilitate access to and ease further mining of the data by the scientific community, all data can be explored online in a purpose-built R Shiny app (https://brain-region-atlas.proteomics.ls.tum.de).

Characterization of Adeno-Associated Virus Capsid Proteins by Microflow Liquid Chromatography Coupled with Mass Spectrometry.

Adeno-associated virus (AAV) has been widely used to treat various human diseases as an important delivery vector for gene therapy due to its low immunogenicity and safety. AAV capsids proteins are comprised of three capsid viral proteins (VP; VP1, VP2, VP3). The capsid proteins play a key role in viral vector infectivity and transduction efficiency. To ensure the safety and efficacy of AAV gene therapy products, the quality of AAV vector capsid proteins during development and production should be carefully monitored and controlled. Microflow liquid chromatography coupled with mass spectrometry provides superior sensitivity and fast analysis capability. It showed significant advantages in the analysis of low- concentration and large numbers of AAV samples. The intact mass of capsid protein can be accurately determined using high-resolution mass spectrometry (MS). And MS also provides highly confident confirmation of sequence coverage and post-translational modifications site identification and quantitation. In this study, we used microflow liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the characterization of AAV2 capsid protein. we obtained nearly 100% sequence coverage of low-concentration AAV2 capsid protein (8 × 1011 GC/mL). More than 30 post-translational modifications (PTMs) sites were identified, the PTMs types included deamidation, oxidation and acetylation. From this study, the proposed microflow LC-MS/MS method provides a sensitive and high throughput approach in the characterization of AAVs and other biological products with low abundance.

Unified Workflow for the Rapid and In-Depth Characterization of Bacterial Proteomes

Bacteria are the most abundant and diverse organisms among the kingdoms of life. Due to this excessive variance, finding a unified, comprehensive, and safe workflow for quantitative bacterial proteomics is challenging. In this study, we have systematically evaluated and optimized sample preparation, mass spectrometric data acquisition, and data analysis strategies in bacterial proteomics. We investigated workflow performances on six representative species with highly different physiologic properties to mimic bacterial diversity. The best sample preparation strategy was a cell lysis protocol in 100% trifluoroacetic acid followed by an in-solution digest. Peptides were separated on a 30-min linear microflow liquid chromatography gradient and analyzed in data-independent acquisition mode. Data analysis was performed with DIA-NN using a predicted spectral library. Performance was evaluated according to the number of identified proteins, quantitative precision, throughput, costs, and biological safety. With this rapid workflow, over 40% of all encoded genes were detected per bacterial species. We demonstrated the general applicability of our workflow on a set of 23 taxonomically and physiologically diverse bacterial species. We could confidently identify over 45,000 proteins in the combined dataset, of which 30,000 have not been experimentally validated before. Our work thereby provides a valuable resource for the microbial scientific community. Finally, we grew Escherichia coli and Bacillus cereus in replicates under 12 different cultivation conditions to demonstrate the high-throughput suitability of the workflow. The proteomic workflow we present in this manuscript does not require any specialized equipment or commercial software and can be easily applied by other laboratories to support and accelerate the proteomic exploration of the bacterial kingdom.

Quantification of Proteins in Blood by Absorptive Microtiter Plate-Based Affinity Purification Coupled to Liquid Chromatography-Mass Spectrometry.

Liquid chromatography (LC) coupled to mass spectrometry (MS) is increasingly used for quantification of proteins in blood. This development is prompted by ongoing improvements in detection sensitivities of LC-MS instruments and corresponding sample preparation workflows. The combination of immunoaffinity enrichment and targeted LC-MS detection is a notable analytical platform in this regard as it allows for the quantification of low abundance proteins in biological matrices like plasma and serum. Here, we describe such hybrid methods which are based on the enrichment of proteins with antibodies or affimers coupled to adsorptive microtiter plates, the proteolytic digestion of enriched proteins to release protein-specific peptides, and the detection of these peptides by microflow LC coupled to selected reaction monitoring MS.

Label-free and de-conjugation-free workflow to simultaneously quantify trace amount of free/conjugated and protein-bound estrogen metabolites in human serum

Different chemical forms of sex hormones including free/conjugated metabolites as well as their protein/DNA adducts in human serum are a panel of important indicators of health conditions. It is, however, hard to quantify all species simultaneously due to the lack of general extraction, derivatization, and de-conjugation methods. Here we developed a label-free and de-conjugation-free workflow to quantify 11 free/conjugated estrogen metabolites including depurinating DNA and protein adduct forms of 4-hydroxyestradiol (4OHE2) in human serum. Acetonitrile acts as an excellent solvent to purify adducted and non-adducted human serum albumin (HSA) by precipitation as well as to extract free/conjugated metabolites and depurinating DNA adducts from the supernatant by salting-out effect. The adduction level of 4OHE2 on HSA was determined by proteomics; free/conjugated metabolites were quantified by a newly developed microflow liquid chromatography (microflow LC)-nanoelectrospray ionization (nanoESI)-multiple reaction monitoring (MRM) method with high reproducibility (7–22% RSD, n > 3) and sub-picogram levels (0.6–20 pg/mL) of quantification limits (S/N = 8) by using non-pulled capillary as nano-ESI emitter. This workflow was demonstrated to reveal endogenous adduction level of 4OHE2 on HSA as well as circulation levels of free/conjugated metabolites in clinical samples. 4OHE2 in human serum were solely detected as protein-bound form, indicating the merit of such integrated platform covering unstable or active metabolites. Compared to traditional methods using labeling or de-conjugation reaction, this workflow is much simplier, more sensitive, and more specific. Moreover, it can be widely applied in omics to concurrently access various bio-transformed known and un-known markers or drugs.

Robust Capillary- and Micro-Flow Liquid Chromatography-Tandem Mass Spectrometry Methods for High-Throughput Proteome Profiling.

Capillary- and micro-flow liquid chromatography-tandem mass spectrometry (capLC-MS/MS and μLC-MS/MS) is becoming a valuable alternative to nano-flow LC-MS/MS due to its high robustness and throughput. The systematic comparison of capLC-MS/MS and μLC-MS/MS systems for global proteome profiling has not been reported yet. Here, the capLC-MS/MS (150 μm i.d. column, 1 μL/min) and μLC-MS/MS (1 mm i.d. column, 50 μL/min) systems were both established based on UltiMate 3000 RSLCnano coupled to an Orbitrap Exploris 240 by integrating with different flowmeters. We evaluated both systems in terms of sensitivity, analysis throughput, separation efficiency, and robustness. capLC-MS/MS was about 10 times more sensitive than μLC-MS/MS at different gradient lengths. Compared with capLC-MS/MS, μLC-MS/MS was able to achieve higher analysis throughput and separation efficiency. During the 7 days' long-term performance test, both systems showed good reproducibility of chromatographic full width (RSD < 3%), retention time (RSD < 0.4%), and protein identification (RSD < 3%). These results demonstrate that capLC-MS/MS is more suitable for high-throughput analysis of clinical samples with a limited starting material. When enough samples are available, μLC-MS/MS is preferred. Together, capLC and μLC coupled to Orbitrap Exploris 240 with moderate sensitivity should well meet the needs of large-cohort clinical proteomic analysis.

Microflow Liquid Chromatography Coupled to Mass Spectrometry (μLC-MS) Workflow for O-Glycopeptides Isomers Analysis Combining Differential Mobility Spectrometry and Collision Induced and Electron Capture Dissociation.

Peptide and protein O-glycosylation can occur mostly on any serine or threonine and could generate several positional isomers, which may coelute during liquid chromatography (LC) separation, challenging their characterization. Ion mobility has emerged as a technique of interest to separate isomeric compounds. In the different ion mobility techniques, differential ion mobility (DMS) includes the particular interest to tune ion separation by the possible addition of an organic modifier. Different microflow liquid chromatography coupled to mass spectrometry (μLC-MS) workflows were investigated for the analysis of a set of four model peptides made of three isomeric glycopeptides and a corresponding nonglycosylated peptide using differential mobility spectrometry (DMS), collision induced dissociation (CID), and electron capture dissociation (ECD). Neither DMS nor LC provided sufficient separation of the three isomeric O-glycopeptides while the nonmodified one was clearly separated by LC. The hyphenation of LC with DMS led to differentiating the three glycopeptides, and further detection and characterization (ECD/CID) with a chimeric collision cell were achieved in a single LC run. The position of the modification was determined from ECD data, while CID data characterized the sugar through its distinctive oxoniums ions in the low mass range.

Multiplexed Assay to Quantify the PP-Fold Family of Peptides in Human Plasma Using Microflow Liquid Chromatography-Tandem Mass Spectrometry.

Peptide Tyr-Tyr (PYY1-36), pancreatic polypeptide (PP1-36) and neuropeptide Y (NPY1-36) constitute the PP-fold family of peptides that is involved in metabolic regulation. Very low plasma concentrations and cleavage into active 3-36 fragments challenge bioanalytical assays used for the quantification of these peptides. We developed a multiplexed isotopic dilution assay to quantify PYY1-36, PP1-36, and NPY1-36 and their dipeptidyl peptidase-4 (DPP4)-derived metabolites PYY3-36, PP3-36 and NPY3-36. All peptides were immunocaptured from plasma using a monoclonal antibody and quantified by micro-ultra-HPLC-MS/MS. Blood samples from healthy volunteers were collected fasting and 30 min after nutrient stimulation. Method comparison was performed with commercial immunoassays. Linearity was shown in the measured intervals (r2 > 0.99). The lower limit of quantification (LLOQ) with a CV at 20% was 1.5 pM for PYY1-36 and PYY3-36, 3.0 pM for PP1-36 and PP3-36, 0.8 pM for NPY1-36 and 0.5 pM for NPY3-36. In all cases, intra- and inter-assay bias and imprecision were <21%. Pre-analytical stability required addition of a protease inhibitor cocktail. Physiological concentrations of PYY3-36, NPY3-36, PP1-36 and PP3-36 were above the LLOQ in 43% to 100% of the samples. PYY1-36 and NPY1-36 were above the LLOQ in 9% and 0% of the samples, respectively. Immunoassays showed higher concentrations of measurands and poor agreement when compared with micro-UHPLC-MS/MS. The assay allowed for specific multiplexed analysis of the PP-fold family of peptides and their DPP4-cleaved fragments in a single sample, thereby offering new perspectives to study the role and therapeutic potential of these essential peptide hormones in health and metabolic disease.

Optimized Targeted Proteomics Methodology for Normalizing Tissue Protein Abundance Data in Analysis of Placental Markers

Objective Placental tissue analysis provides important information about the in-utero environment that could have an impact on fetal development. Due to the heterogeneity and variability of perfusion in placental tissue, the technical variability during sample collection can confound placental proteomics data analysis. The aim of this study was to develop a targeted proteomics method for selecting housekeeping placental proteins for normalizing placental data. Methods A list of placental housekeeping proteins was selected based on previously reported placental house-keeping genes (mRNA) (1). The surrogate peptides of the housekeeping proteins were selected for targeted analysis based on an optimized strategy (2). Placental tissue (n=5) was collected from women enrolled in the CANDLE (Conditions Affecting Neurocognitive Development and Learning in Early childhood) study. The samples were fractionated into cytosolic and membrane fractions, and the fractions were enriched, reduced, alkylated, and digested based on previously reported methods (2). Targeted proteomics data were acquired on Waters XevoTQ-XS with IonKey and a microflow liquid chromatography in multiple reaction monitoring mode. The results were analyzed using Skyline software. The coefficient of variation (%CV) of the proteins was calculated to determine the technical or inter-individual variability. Results The following surrogate peptides of the housekeeping proteins were reproducibly quantified: VIGGDDLSTLTGK (HPRT); LGANSLLDLVVFGR (SDHA); LGFQVIFTDFK (TBPL1); DSTLIMQLLR (1433Z); DLLHVLAFSK (LEP); GPQLAAQNLGISLANLLLSK (HEM3); VIHDNFGIVEGLMTTVHAITATQK (G3P); SVALAVLALLSLSGLEAIQR (B2MG); NLVQEWLAK (PPB1); VFDEFKPLVEEPQNLIK (ALBUMIN); and IVEIPFNSTNK (AT1A1), with the fragments and peptides correlation (R2) equal to 0.8-0.99 and 0.80-0.96, respectively. HPRT, SDHA, 1433Z, HEM3, G3P, and B2MG were enriched in the cytosolic fraction, whereas only PPB1 and AT1A1 were enriched in the membrane fraction. TBPL1 and LEP were detected in both fractions. The following proteins showed %CV below 20%: HPRT, SDHA, TBPL1, HEM3, and AT1A1. Positive correlation was observed between 1433Z and G3P, and HPRT and B2MG with an R2 equal to 0.98 and 0.86, respectively. HPRT and SDHA were negatively correlated with an R2 equal to 0.95. Discussion We confirmed that housekeeping proteins could be quantified with good correlation and low coefficients of variation. The placenta is an important organ and its research applications in birth cohort-based studies requires consistent data normalization techniques. Proteomics analysis of placental tissue is paramount to understand the placental phenotype since the proteome is closely associated with function. Creating a cocktail method of housekeeping protein markers that can be used to normalize the protein concentration is vital to comparing samples and data within and across laboratories.

Robust Microflow LC-MS/MS for Proteome Analysis: 38 000 Runs and Counting.

Microflow liquid chromatography tandem mass spectrometry (μLC-MS/MS) is becoming a viable alternative to nanoflow LC-MS/MS for the analysis of proteomes. We have recently demonstrated the potential of such a system operating with a 1 mm i.d. × 150 mm column and at a flow rate of 50 μL/min for high-throughput applications. On the basis of the analysis of ∼38 000 samples measured on two instruments over the past two years, we now show that the approach is extremely robust. Up to 1500 analyses were performed within one month, and >14 000 samples could be analyzed on a single column without loss of chromatographic performance. Samples included proteomes of cell lines, tissues, and human body fluids, which were analyzed with or without prior peptide fractionation or stable isotope labeling. We show that the μLC-MS/MS system is capable of measuring 2600 proteins from undepleted human plasma and ∼5000 proteins from crude human urine in 1 day, demonstrating its potential for in-depth as well as high-throughput clinical application.

Abstract PS4-48: Development of a multiplexed protein panel using a targeted proteomics approach for the study of CDK4/6 inhibitors resistance in hormone receptor positive breast cancer

Abstract Background Breast cancer is the most common malignancy in woman and hormone receptor positive breast cancer represents approximately 70% of all cases. These patients are treated with endocrine therapies aimed at disrupting hormone receptor signaling and estrogen production which improves survival and allows a cure in early stages. However, recurrent disease, metastatic dissemination and drug resistance limit the survival of patients. The limitations regarding endocrine therapy have prompted the search for new therapeutic targets, such as CDK4/6 Inhibitors. Despite the improved disease control that CDK4/6 Inhibitors offer to patients, not all patients respond to these drugs and some patients whose tumors respond to CDK4/6 Inhibitors eventually develop acquired resistance. No proven biomarkers of CDK4/6 Inhibitors efficacy exist to date, and there is a need for diagnostic tools that could stratify patients to save costs and the burden of unnecessary therapy. Our aim is to perform a quantitative evaluation of marker proteins with a developed multiplexed panel using targeted mass spectrometry (MS)-based proteomics for 25 proteins from the CDK/RB/E2F-pathway which have been shown in the literature to be central to CDK4/6I resistance. Material and Methods We developed Multiple Reaction Monitoring (MRM) MS methods for the 25 target proteins from the CDK/RB/E2F-pathway using synthetic heavy-isotope-labeled standards with the aim of creating MRM assays to enable specific, sensitive and precise quantitation of these proteins in small amounts of cell line and tissue samples. Moreover, we developed a high resolution peptide fractionation system using high-pH micro-flow liquid chromatography (LC) which is required to overcome the problem of small samples amounts while improving analytical assay sensitivity in the analysis of complex biological matrices such as breast cancer biopsies. The MCF-7 human breast cancer cell line was used as model during method development. Proteins from cell lysates were isolated, reduced, alkylated and digested with trypsin. The resulting tryptic peptides were micro-flow fractionated into 70 fractions and the developed nano-LC-MS MRM assays were used for peptide detection and quantification. Data were analyzed using Skyline. Results Our developed micro-flow fractionation method allowed us to work on limited amounts of samples (60ug), and increased the possibility to detecting low abundance proteins such as cell cycle components. Using the MCF-7 cell model, we are able to identify and quantify 17 proteins out of the 25 from our panel: CDK1, CDK2, CDK4, Cyclin B1, Cyclin D1, Cyclin D3, Cyclin E1, RB1, E2F-3, E2F-4, E2F-5, ESR1, TOP2A, TYMS, EZH2, MKI67, BIRC5. Conclusion We have developed a highly specific MS-based multiplexed assay with peptide standards targeting 25 proteins relevant to CDK4/6 Inhibitors breast cancer treatment. Our micro-flow fractionation method increased assay sensitivity and allows for the analysis of small sample amounts. In the future we will apply this workflow to samples such as Patient Derived Xenografts models, breast cancer tissues and FFPE samples in order to identify the predictive value of these potential biomarkers for responsiveness to CDK4/6 Inhibitors. Citation Format: Marta Zurawska, Adriana Aguilar-Mahecha, Mark Basik, Michal Dadlez, Dominik Domanski. Development of a multiplexed protein panel using a targeted proteomics approach for the study of CDK4/6 inhibitors resistance in hormone receptor positive breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-48.

Combining Field-Enabled Capillary Vibrating Sharp-Edge Spray Ionization with Microflow Liquid Chromatography and Mass Spectrometry to Enhance 'Omics Analyses.

Field-enabled capillary vibrating sharp-edge spray ionization (cVSSI) has been combined with high-flow liquid chromatography (LC) and mass spectrometry (MS) to establish current ionization capabilities for metabolomics and proteomics investigations. Comparisons are made between experiments employing cVSSI and a heated electrospray ionization probe representing the state-of-the-art in microflow LC-MS methods for 'omics studies. For metabolomics standards, cVSSI is shown to provide an ionization enhancement by factors of 4 ± 2 for both negative and positive ion mode analyses. For chymotryptic peptides, cVSSI is shown to provide an ionization enhancement by factors of 5 ± 2 and 2 ± 1 for negative and positive ion mode analyses, respectively. Slightly broader high-performance liquid chromatography peaks are observed in the cVSSI datasets, and several studies suggest that this results from a slightly decreased post-split flow rate. This may result from partial obstruction of the pulled-tip emitter over time. Such a challenge can be remedied with the use of LC pumps that operate in the 10 to 100 μL·min-1 flow regime. At this early stage, the proof-of-principle studies already show ion signal advantages over state-of-the-art electrospray ionization (ESI) for a wide variety of analytes in both positive and negative ion mode. Overall, this represents a ∼20-50-fold improvement over the first demonstration of LC-MS analyses by voltage-free cVSSI. Separate comparisons of the ion abundances of compounds eluting under identical solvent conditions reveal ionization efficiency differences between cVSSI and ESI and may suggest varied contributions to ionization from different physicochemical properties of the compounds. Future investigations of parameters that could further increase ionization gains in negative and positive ion mode analyses with the use of cVSSI are briefly presented.

The separation and identification of synthetic cathinones by portable low microflow liquid chromatography with dual capillary columns in series and dual wavelength ultraviolet detection.

This study ascertained the viability of a portable liquid chromatograph, operating at low microliter per minute flow, for the analysis of seized drugs at remote sites as well as in laboratory settings. Synthetic cathinones were screened using dual capillary columns in series, C8 and biphenyl, with on-column ultraviolet detection at 255 and 275nm. The relative retention times of the two columns in series and their peak area absorbance ratio were used to determine if the 16 synthetic cathinones investigated could be uniquely identified in these conditions. Based on these parameters all of the analytes could be distinguished. Representative mixtures of synthetic cathinones were used to determine the repeatability, linearity, and limits of detection of the method. This cost effective and green instrumentation has the potential to satisfy minimum international guidelines for the analysis of seized drugs.

Targeted mass spectrometry of ketamine and its metabolites cis-6-hydroxynorketamine and norketamine in human blood serum

Ketamine (KET) was originally developed as an anesthetic agent but has also attracted attention for further clinical applications such as medical treatment of depression or pain. The use of KET induces dissociation and emergence delirium. Due to these effects, KET has a high potential for abuse. In order to investigate metabolization of KET or to confirm misuse of KET, highly sensitive analytical methods that cover KET and its metabolites are necessary. A new analytical approach for simultaneous analysis of KET and its metabolites cis-6-hydroxynorketamine (HNK) and norketamine (NK) was established. The compounds were extracted from human blood serum by ultrafiltration and solid phase extraction with subsequent vacuum evaporation. The compounds were analyzed by non-enantioselective ultra-high performance micro-flow liquid chromatography (Waters ACQUITY UPLC® M-Class HSS T3 column, 0.1% formic acid and acetonitrile with 0.1% formic acid, 14 µL/min flow rate) coupled with tandem mass spectrometry in positive scheduled multiple reaction monitoring mode. Validation parameters such as linearity, precision, recovery, accuracy, stability, limit of detection (LOD), and limit of quantification (LOQ) were proven. LOD for KET and NK was 0.08 ng/mL and LOQ were 0.5 ng/mL and 0.6 ng/mL, respectively. For HNK, LOD was 0.1 ng/mL and LOQ 0.8 ng/mL. The method was then successfully applied to quantify KET, HNK, and NK in blood serum samples from subjects who received KET intravenously. A novel method for the simultaneous analysis of KET, NK, and HNK was established. This new method could now be used for clinical trials investigating KET and its metabolites HNK and NK or for forensic analysis in order to confirm KET abuse.

Accelerating SWATH® Acquisition for Protein Quantitation – Up to 100 Samples per Day

The combination of microflow LC with SWATH Acquisition for large scale quantitative proteomics studies is becoming increasingly more widespread, due to the improved robustness and throughput obtained relative to the traditional nanoflow LC approach. High quality quantitative datasets have been generated using a standard 1 hour gradient, demonstrating large numbers of proteins quantified routinely.The impact of gradient length on protein identification results using data dependent acquisition (DDA) was also explored previously and summarized here. Next, an exploration into the impact of gradient length on proteins quantified using data independent acquisition (DIA) was undertaken to provide researchers expanded workflow options with microflow SWATH acquisition. Using microflow liquid chromatography gradients as short as five minutes (total run time &lt;15 mins), SWATH acquisition parameters were first optimized then used on multiple TripleTOF® 6600 systems to study impact of shortened separations on number of proteins quantified.Testing on multiple instruments with different complex matrices, it was observed that the number of proteins quantified using SWATH acquisition for the fastest gradients was over 1300 proteins for multiple complex matrices from 1 μg protein load. When combined with larger ion libraries, over 2100 proteins were identified and quantified in a 5 min gradient from a complex HEK digest (1 μg). The combination of microflow LC with the high MS/MS acquisition rates of the TripleTOF® 6600 system enables high‐throughput yet comprehensive analysis for proteomics samples, at rates approaching 100 samples per day.

Blood mass spectrometry detects residual disease better than standard techniques in light-chain amyloidosis

In patients with immunoglobulin light-chain (AL) amyloidosis, depth of hematologic response correlates with both organ response and overall survival. Our group has demonstrated that screening with a matrix-assisted laser desorption/ionization-time-of-flight (TOF) mass spectrometry (MS) is a quick, sensitive, and accurate means to diagnose and monitor the serum of patients with plasma cell disorders. Microflow liquid chromatography coupled with electrospray ionization and quadrupole TOF MS adds further sensitivity. We identified 33 patients with AL amyloidosis who achieved amyloid complete hematologic response, who also had negative bone marrow by six-color flow cytometry, and who had paired serum samples to test by MS. These samples were subjected to blood MS. Four patients (12%) were found to have residual disease by these techniques. The presence of residual disease by MS was associated with a poorer time to progression (at 50 months 75% versus 13%, p = 0.003). MS of the blood out-performed serum and urine immunofixation, the serum immunoglobulin free light chain, and six-color flow cytometry of the bone marrow in detecting residual disease. Additional studies that include urine MS and next-generation techniques to detect clonal plasma cells in the bone marrow will further elucidate the full potential of this technique.