Immunodepletion of high abundance proteins coupled on-line with reversed-phase liquid chromatography: a two-dimensional LC sample enrichment and fractionation technique for mammalian proteomics.

Proteomic analysis can be hampered by the large concentration distribution of proteins. Immunoaffinity techniques have been applied to selectively remove high abundant proteins (HAP's) from samples prior to analysis. Although immunodepletion of HAP's has been shown to enable greater detection of low abundance proteins, the resulting fractions are often diluted 5-10-fold during the process. Various concentration techniques can be applied; however, many are incompatible with the high salt content of the fractions. To help overcome this limitation, a two-dimensional liquid chromatography (2D-LC) method was developed which couples an IgY immunodepletion column in the first dimension with a large pore C18 analytical column in the second. A protein trap cartridge serves as an injection loop between the columns to facilitate on-line concentration and desalting. Feasibility of this 2D-LC system was demonstrated for mammalian proteomics. Beyond depletion of interfering proteins, this instrumentation provides four advantages which make immunodepletion technology more convenient, including: (1) on-line desalting (2) automatic buffer exchange (3) facile concentration and (4) fractionation by polarity.

Cross species applicability of abundant protein depletion columns for ribulose-1,5-bisphosphate carboxylase/oxygenase.

In plants, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is an important enzyme in the Calvin cycle, catalyzing the first step of carbon fixation. Because of its critical role in photosynthesis, RuBisCO comprises 30-60% of the total protein content in green leaf tissue and represents a major protein which can interfere with determination of lower abundance proteins in plant proteomics. A potential solution to aid in the determination of low level proteins in plant proteomics are RuBisCO immunodepletion columns. Two formats, spin and LC, of Seppro IgY RuBisCO depletion columns were evaluated for cross species applicability. The spin and LC columns were found to deplete arabidopsis RuBisCO by greater than 90 and 98%, respectively, and automation could be achieved with the LC format. Canola RuBisCO was depleted to a similar extent, and there was evidence suggesting that corn and tobacco RuBisCO were also highly depleted in flow through fractions. Model proteins were spiked into samples to provide insight into the degree of non-specific binding. Finally, improved detection and identification of lower abundance proteins was demonstrated after depletion.

Microdialysis coupled on-line to capillary liquid chromatography with tandem mass spectrometry for monitoring acetylcholine in vivo.

Capillary liquid chromatography-mass spectrometry (cLC-MS) was coupled on-line to microdialysis sampling to monitor endogenous acetylcholine (ACh) from the rodent brain. In vivo microdialysate sampled at 0.6 microL/min from the striatum of ketamine or chloral hydrate anesthetized rats was loaded onto a sample loop and then injected onto a approximately 5 cm long strong cation exchange (SCX) capillary column. A step gradient was used to separate the analyte from ionization suppressing salts contained in dialysate in 2.4 min. Sampling coupled on-line with cLC-MS allowed for high temporal resolution (data points at 2.4 min intervals), good reproducibility (10-15% relative standard deviation, R.S.D.), and sensitive limits of detection (0.04 nM or 8 amol injected). The method successfully monitored basal and stimulated levels (induced by increased K+ concentrations) of ACh from the anesthetized rat without necessitating perfusion of an acetylcholinesterase (AChE) inhibitor. Absolute and percent basal levels of ACh from rats receiving different anesthetics were also compared.

A capillary-PDMS hybrid chip for separations-based sensing of neurotransmitters in vivo.

A chip fabricated by multilayer soft lithography of poly(dimethylsiloxane) was created for separations-based sensing of neurotransmitters in vivo. The chip incorporated a pneumatically actuated peristaltic pump and valving system to combine low-flow push-pull perfusion sampling, on-line derivatization, and flow-gated injection onto an embedded fused-silica capillary for high speed separation of amine neurotransmitters from the brain of living animals. Six 160 microm wide by 10 microm high control channels, actuated with an overlapping 60 degrees pulse sequence, simultaneously drove sample and buffers through fluidic channels of the same dimensions. Tunable sampling flow rates of 40 to 130 nL min(-1) and separation buffer flow rates of 380 to 850 nL min(-1) were achieved with actuation frequencies between 3 and 10 Hz. On-line sampling of amine neurotransmitters with separation efficiencies in excess of 250,000 plates, detection limits of approximately 40 nM, and relative standard deviations of 4% for glutamate and aspartate were achieved in vitro. Electropherograms with resolution of gamma-aminobutyric acid, glutamine, taurine, serine, glycine, o-phosphorylethanolamine, glutamate, and aspartate could be collected every 30 s for over 4 h in vivo. It was also shown that pharmacological agents could be delivered and subsequent changes in neurotransmitter profile could be measured when delivering either 70 mM K+ artificial cerebrospinal fluid or 200 microM l-trans-pyrrolidine-2,4-dicarboxilic acid with the chip. These results demonstrate the ability of this chip to sample and monitor chemicals in the complex environment of the central nervous system with high selectivity and sensitivity over extended periods.

Simultaneous Determination of Total Vitamins B1, B2, B3, and B6 in Infant Formula and Related Nutritionals by Enzymatic Digestion and LC-MS/MS: Single-Laboratory Validation, First Action 2015.14.

This method provides simultaneous determination of total vitamins B1, B2, B3, and B6 in infant formula and related nutritionals (adult and infant). The method was given First Action for vitamins B1, B2, and B6, but not B3, during the AOAC Annual Meeting in September 2015. The method uses acid phosphatase to dephosphorylate the phosphorylated vitamin forms. It then measures thiamine (vitamin B1); riboflavin (vitamin B2); nicotinamide and nicotinic acid (vitamin B3); and pyridoxine, pyridoxal, and pyridoxamine (vitamin B6) from digested sample extract by liquid chromatography-tandem mass spectrometry. A single-laboratory validation was performed on 14 matrixes provided by the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) to demonstrate method effectiveness. The method met requirements of the AOAC SPIFAN Standard Method Performance Requirement for each of the three vitamins, including average over-spike recovery of 99.6 ± 3.5%, average repeatability of 1.5 ± 0.8% relative standard deviation, and average intermediate precision of 3.9 ± 1.3% relative standard deviation.

A new sample preparation and separation combination for precise, accurate, rapid, and simultaneous determination of vitamins B1, B2, B3, B5, B6, B7, and B9 in infant formula and related nutritionals by LC-MS/MS.

An improved method was developed for simultaneous determination of the fortified forms of thiamine (B1), riboflavin (B2), nicotinamide and nicotinic acid (B3), pantothenic acid (B5), pyridoxine (B6), biotin (B7), and folic acid (B9) in infant formulas and related nutritionals. The method employed a simple, effective, and rapid sample preparation followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). It improved upon previous methodologies by offering facile and rugged sample preparation with improved chromatographic conditions, which culminated in a highly accurate and precise method for water-soluble vitamin determination in a wide range of formulas. The method was validated over six days in ten unique matrices with two analysts and on instruments in two different labs. Intermediate precision averaged 3.4 ± 2.6% relative standard deviation and over-spike recovery averaged 100.2 ± 2.4% (n = 160). Due to refinements in sample preparation, the method had high sample throughput capacity.

Capillary liquid chromatography with MS3 for the determination of enkephalins in microdialysis samples from the striatum of anesthetized and freely-moving rats.

In vivo microdialysis sampling was coupled to capillary liquid chromatography (LC)/electrospray ionization quadrupole ion trap mass spectrometry (MS) to monitor [Met]enkephalin and [Leu]enkephalin in the striatum of anesthetized and freely-moving rats. The LC system utilized a high-pressure pump to load 2.5 microl samples and desalt the 25 microm i.d. by 2 cm long column in 12 min. Samples were eluted with a separate pump at approximately 100 nl min(-1). A rapid gradient effectively separated the endogenous neuropeptides in 4 min. A comparison was made for operating the mass spectrometer in the MS2 and MS3 modes for detection of the peptides. In standard solutions, the detection limits were similar at 1-2 pM (2-4 amol injected); however, the reproducibility was improved with MS3 as the relative standard deviation was <5% compared with 20% for MS2 for 60 pM samples. For dialysate solutions, reconstructed ion chromatograms and tandem mass spectra had much higher signal-to-noise ratios in the MS3 mode, resulting in more confident detection at in vivo concentrations. The method was successfully used to monitor the peptides under basal conditions and with stimulation of peptide secretion by infusion of elevated K+ concentration.

Microfluidic chip for low-flow push-pull perfusion sampling in vivo with on-line analysis of amino acids.

Multilayer soft lithography was used to prepare a poly(dimethylsiloxane) microfluidic chip that allows for in vivo sampling of amino acid neurotransmitters by low-flow push-pull perfusion. The chip incorporates a pneumatically actuated peristaltic pump to deliver artificial cerebrospinal fluid to a push-pull perfusion probe, pull sample from the probe, perform on-line derivatization with o-phthaldialdehyde, and push derivatized amino acids into the flow-gated injector of a high-speed capillary electrophoresis-laser-induced fluorescence instrument. Peristalsis was achieved by sequential actuation of six, 200 microm wide by 15 microm high control valves that drove fluid through three fluidic channels of equal dimensions. Electropherograms with 100,000 theoretical plates were acquired at approximately 20-s intervals. Relative standard deviations of peak heights were 4% in vitro, and detection limits for the excitatory amino acids were approximately 60 nM. For in vivo measurements, push-pull probes were implanted in the striatum of anesthetized rats and amino acid concentrations were monitored while sampling at 50 nL/min. o-Phosphorylethanolamine, glutamate, aspartate, taurine, glutamine, serine, and glycine were all detected with stable peak heights observed for over 4 h with relative standard deviations of 10% in vivo. Basal concentrations of glutamate were 1.9 +/- 0.6 microM (n = 4) in good agreement with similar methods. Monitoring of dynamic changes of neurotransmitters resulting from 10-min applications of 70 mM K(+) through the push channel of the pump was demonstrated. The combined system allows temporal resolution for multianalyte monitoring of approximately 45 s with spatial resolution 65-fold better than conventional microdialysis probe with 4-mm length. The system demonstrates the feasibility of sampling from a complex microenvironment with transfer to a microfluidic device for on-line analysis.

Discovery and neurochemical screening of peptides in brain extracellular fluid by chemical analysis of in vivo microdialysis samples.

Endogenous peptides from brain extracellular fluid of live rats were analyzed using capillary liquid chromatography (LC)-tandem mass spectrometry (MS2). A 4-mm-long microdialysis probe perfused at 0.6 microL/min implanted into the striatum of anesthetized male rats was used to collect 3.6 microL dialysate fractions that were injected on-line into the capillary LC-MS2 system for analysis. A total of 3349 MS2 spectra were collected from 13 different animals under basal conditions and during localized depolarization evoked by infusion of a high-K+ solution through the microdialysis probe. Subtractive analysis revealed a total of 859 MS2 spectra that were observed only during depolarization. From these spectra, 29 peptide sequences (25 were peptides not previously observed) from 6 different protein precursors were identified using database searching software. Proteins identified include precursors to neuropeptides, synaptic proteins, blood proteins, and transporters. The identified peptides represent candidates for neurotransmitters, neuromodulators, and markers of synaptic activity or brain tissue damage. A screen for neuroactivity of novel proenkephalin fragments that were found was performed by infusing the peptides into the brain while monitoring amino acid neurotransmitters by microdialysis sampling combined with capillary electrophoresis. Three of the six tested peptides evoked significant increases in various neuroactive amino acids. These results demonstrate that this combination of methods can identify novel neurotransmitter candidates and screen for potential neuroactivity.