Application of zero-phase digital filtering for effective denoising of field asymmetric waveform ion mobility spectrometry signal.

RATIONALE: Field asymmetric waveform ion mobility spectrometry (FAIMS) has a great potential to become a portable technology for rapid detection of chemical and biological agents. However, the ion current signals, measured at the exit of the planar FAIMS directly, may contain different types of noises. The peak information in the FAIMS spectrum, such as the compensation voltage (CV) value at the maximum peak intensity (CVP ) and the peak width at half maximum (Wh ), could not be accurately determined under the weak signal condition, which significantly limits the achievable instrument sensitivity, and there are no existing solutions to the problem. METHODS: This study analyzed the noise type of FAIMS signal in detail, and three different signal processing algorithms, such as median filtering (MF), discrete wavelet transform (DWT), and zero-phase digital filtering (ZDF), were evaluated for their performance in denoising the FAIMS signal. RESULTS: The results show that the standard deviation of CVp obtained from the signal denoised using ZDF algorithm is at least 31.82% smaller as compared to using MF and DWT algorithms. The standard deviation of Wh is at least 45.45% smaller using ZDF algorithm. Moreover, only ZDF algorithm can keep the percentage error for the CV value of the denoised signal to be within 0.50 ± 0.47% of the true CV value, implying the effectiveness of ZDF algorithm in denoising while retaining the integrity of the signal. CONCLUSIONS: The ZDF algorithm greatly reduces the analyte peak extraction error and improves the limit of detection in FAIMS measurements.

Hollow electrode capillary plasma ionization source for rapid online detection of gaseous samples.

RATIONALE: Gas chromatography mass spectrometry (GC-MS) with electron ionization (EI) is the most widely used analysis technique of gaseous samples, but it may be time-consuming for online monitoring of mixtures whose concentrations relatively change rapidly. On the contrary, current ionization methods, such as chemical ionization (CI) and proton transfer reaction (PTR), also have some disadvantages such as selectivity. Therefore, appropriate soft ionization sources are searched for rapid online detection. METHODS: Hollow electrode capillary plasma ionization (HECPI) is based on single electrode plasma. A hollow capillary was placed as both the electrode and the inlet of the gaseous samples. The ionization source is coupled with a mass spectrometer for performance evaluation. RESULTS: Several typical compounds have been tested with HECPI-mass spectrometer. In this process, the dominant ion peaks of all compounds can be indexed as molecular ion peaks, and the product ions of HECPI are less than that of dielectric barrier discharge ionization (DBDI). Three gaseous samples (linalool, triethylamine, and styrene) with various concentrations have been used to further confirm the performance of this source, and the detection limit of linalool is as low as 10 ppb. CONCLUSIONS: HECPI is simple in structure and shows good performance. The results also show that HECPI has the potential to be an effective tool for detecting online gaseous samples rapidly.

Monodisperse microsphere-based immobilized metal affinity chromatography approach for preparing Antarctic krill phospholipids followed by HILIC-MS analysis.

Phospholipids enriched krill is a functional food beneficial in cardiovascular diseases. Herein, monodisperse microsphere-based immobilized metal affinity chromatographic material (MM-IMAC) was synthesized with Ti4+ incorporated to enrich phospholipids from krill by coordination with phosphate group. The extract was profiled by hydrophilic interaction chromatography-mass spectrometry (HILIC-MS) with 154 phospholipid molecular species detected. The parameters were loading solvent n-hexane/isopropanol (2:8, v/v), flow rate 0.8 mL·min-1, and eluting volume 1 mL. Besides, eicosapentaenoic and docosahexaenoic acids structured phospholipids were located, such as phosphatidylcholine (PC) 20:5/22:6, phosphatidylinositol (PI) 18:0/20:5, etc. Finally, this method was validated in linearity (R2 ≥ 0.9953), sensitivity (LOD ≤ 0.53 µg·mL-1 and LOQ ≤ 1.66 µg·mL-1), precision (RSDintraday ≤ 4.86% and RSDinterday ≤ 6.25%), and recovery (58-83%). It indicated that the MM-Ti4+-IMAC-HILIC-MS was reliable and efficient in specific study of phospholipids in food matrix.

On a separation voltage polarity switching transient capillary isotachophoresis method for higher sample loading capacity and better separation performance.

Limited sample loading capacity is one of the major reasons that prevents the utility of capillary electrophoresis (CE) as a routine separation method as compared to liquid chromatography (LC). In our previous study, separation voltage polarity switching transient capillary isotachophoresis (PS-tCITP) was proposed. Both sample loading capacity and separation resolution could be improved using a single PS-tCITP instead of routine transient capillary isotachophoresis (tCITP). In this study, a detailed investigation on the optimization strategy of the PS-tCITP method was performed systematically. A possible mechanism of sample preconcentration in multiple PS-tCITP was first proposed to better understand the multiple PS-tCITP process. Several optimization experiments were then performed, including single PS-tCITP, paused PS-tCITP and multiple PS-tCITP, sequentially using a mixture of five peptides. By selecting an optimum polarity switching time, sample loading capacity of 100% capillary volume could be achieved in a single PS-tCITP. Introducing an additional pause between each polarity switching in a single PS-tCITP further improved the separation resolution. Experimental results showed a baseline separation of five selected peptide standards at 100% sample loading volume using a 100 min pause in a single PS-tCITP. To further improve separation efficiency while still maintaining 100% sample loading volume, a multiple PS-tCITP technique was developed through this study. Compared to the separation performance of the optimal single PS-tCITP at 100% sample loading volume with a 10 min pause, the separation window was improved by 54% and the peak capacity was improved by 48% in the optimal four PS-tCITP with the same sample loading volume and pause.

Performance optimization of an ion funnel driven by novel radiofrequency waveforms.

RATIONALE: In mass spectrometry, ion transmission is usually achieved by driving an ion funnel with a reversed sine wave radiofrequency. However, the mass range of this conventional ion funnel is limited. In order to overcome this limitation, and to improve the transmission efficiency of the ion funnel, we explore the use of different radiofrequency waveforms for different m/z ranges. METHODS: Right triangle, sawtooth, and variable phase sine (VPS) waves are used in different m/z ranges to improve ion transmission efficiency. We use SIMION-based numerics to simulate their potential field distributions and ion flight trajectories. We compare transmission and focusing performances with those of a conventional ion funnel. RESULTS: Ions with high m/z values require a larger potential gradient to limit their flight trajectory. Right triangle waves can quickly adjust electrode potential through a step change. The equipotential line distribution of VPS waves is wider than that of a sine wave which improves focusing performance. At the same time, the local ion trap effect at the outlet of the ion funnel is improved because of the "snake potential". The maximum effective potential of the sawtooth wave is smaller than that of the sine wave, which is suitable for low m/z ion transmission. CONCLUSIONS: Sawtooth and VPS waves may improve the transmission performance of the ion funnel in the low m/z regime whereas right triangle waves may improve the transmission performance in the high m/z regime.

In situ synthesis of a novel metal oxide affinity chromatography affinity probe for the selective enrichment of low-abundance phosphopeptides.

RATIONALE: Due to the dynamic nature of phosphorylation states and the low stoichiometry of phosphopeptides, it is still a challenge to efficiently capture phosphopeptides from complex biological samples before mass spectrometry analysis. Among the enrichment strategies, metal oxide affinity chromatography (MOAC) is one of the most widely used and the one with the most potential. It is based on reversible Lewis acid-base interactions between the metal oxides and the negatively charged phosphate groups to achieve the specific selection of phosphopeptides. METHODS: A novel MOAC affinity probe, denoted as G@PDA@ZrO2 , was successfully synthesized by in situ grafting ZrO2 onto the surface of graphene (G) modified with polydopamine (PDA). The novel MOAC probe thus obtained was used for phosphopeptide enrichment. RESULTS: This novel MOAC affinity probe when used to selectively enrich phosphopeptides from standard protein digest solutions exhibited a high selectivity (ß-casein:bovine serum albumin = 1:1000), a low detection limit (4 fmol), and a high loading capacity (400 mg/g). At the same time, the experimental results proved that G@PDA@ZrO2 had great recyclability (five cycles), stability, and reproducibility. Subsequently, G@PDA@ZrO2 was applied to enrich phosphopeptides from human saliva and human serum, in which 25 and 4 phosphopeptide peaks, respectively, were detected. CONCLUSIONS: This novel MOAC affinity probe (G@PDA@ZrO2 ) showed good performance in enriching phosphopeptides. Thus, G@PDA@ZrO2 has good potential in phosphopeptidomics analysis.

A separation voltage polarity switching method for higher sample loading capacity and better separation resolution in transient capillary isotachophoresis separation.

A separation voltage polarity switching transient capillary isotachophoresis (PS-tCITP) was developed to overcome a major sample loading volume limitation in transient capillary isotachophoresis (tCITP). The fundamental idea of PS-tCITP is to let sample ions move back and forth in a separation capillary during their initial isotachophoresis focusing stage by switching the polarity of the separation voltage, in order to both increase the sample loading volume and improve the separation efficiency as compared to the conventional tCITP method. The experimental evaluation of the novel PS-tCITP method by using two peptide standards at 2 µM concentration showed that the maximum sample loading volume could be increased from 45% of the total separation capillary volume in tCITP to 70% in PS-tCITP, which resulted in a more than 1.5 fold increase in the peptide peak intensity at a given length/volume of the separation capillary. Due to the consecutive focusing of sample volume from each polarity switching of the separation voltage, the separation time window at a given sample loading volume was also increased significantly in PS-tCITP as compared to tCITP. Experiment comparison between tCITP and PS-tCITP at 45% sample loading volume using the same setup showed that the migration time difference between the two peptide peaks increased from 0.3 min in tCITP to 0.363 min in PS-tCITP with similar peak widths and heights, resulting in roughly a 21% improvement in separation resolution. The performance advantages of PS-tCITP separation over tCITP separation were further verified by using a mixture of six peptide standards.

Carrier-Assisted Single-Tube Processing Approach for Targeted Proteomics Analysis of Low Numbers of Mammalian Cells.

Heterogeneity in composition is inherent in all cell populations, even those containing a single cell type. Single-cell proteomics characterization of cell heterogeneity is currently achieved by antibody-based technologies, which are limited by the availability of high-quality antibodies. Herein we report a simple, easily implemented, mass spectrometry (MS)-based targeted proteomics approach, termed cLC-SRM (carrier-assisted liquid chromatography coupled to selected reaction monitoring), for reliable multiplexed quantification of proteins in low numbers of mammalian cells. We combine a new single-tube digestion protocol to process low numbers of cells with minimal loss together with sensitive LC-SRM for protein quantification. This single-tube protocol builds upon trifluoroethanol digestion and further minimizes sample losses by tube pretreatment and the addition of carrier proteins. We also optimized the denaturing temperature and trypsin concentration to significantly improve digestion efficiency. cLC-SRM was demonstrated to have sufficient sensitivity for reproducible detection of most epidermal growth factor receptor (EGFR) pathway proteins expressed at levels ≥30 000 and ≥3000 copies per cell for 10 and 100 mammalian cells, respectively. Thus, cLC-SRM enables reliable quantification of low to moderately abundant proteins in less than 100 cells and could be broadly useful for multiplexed quantification of important proteins in small subpopulations of cells or in size-limited clinical samples. Further improvements of this method could eventually enable targeted single-cell proteomics when combined with either SRM or other emerging ultrasensitive MS detection.

Analysis and design of a 3rd order velocity-controlled closed-loop for MEMS vibratory gyroscopes.

The time-average method currently available is limited to analyzing the specific performance of the automatic gain control-proportional and integral (AGC-PI) based velocity-controlled closed-loop in a micro-electro-mechanical systems (MEMS) vibratory gyroscope, since it is hard to solve nonlinear functions in the time domain when the control loop reaches to 3rd order. In this paper, we propose a linearization design approach to overcome this limitation by establishing a 3rd order linear model of the control loop and transferring the analysis to the frequency domain. Order reduction is applied on the built linear model's transfer function by constructing a zero-pole doublet, and therefore mathematical expression of each control loop's performance specification is obtained. Then an optimization methodology is summarized, which reveals that a robust, stable and swift control loop can be achieved by carefully selecting the system parameters following a priority order. Closed-loop drive circuits are designed and implemented using 0.35 µm complementary metal oxide semiconductor (CMOS) process, and experiments carried out on a gyroscope prototype verify the optimization methodology that an optimized stability of the control loop can be achieved by constructing the zero-pole doublet, and disturbance rejection capability (D.R.C) of the control loop can be improved by increasing the integral term.

Expression of survivin in human non-Hodgkin lymphoma and its correlation with proliferation and angiogenesis.

In order to investigate the expression change of survivin in non-Hodgkin lymphoma (NHL) and its possible effects on NHL development, the expression of survivin, Ki-67, caspase3 and FVIIIRAg in reactive lymphoid hyperplasia (RH) and NHL was detected by immunohistochemical assay, and apoptosis index (AI) in RH and NHL by TUNEL analysis. The results showed that the expression of survivin is significantly higher in aggressive NHL than in indolent NHL (P<0.01), while there was no statistically significant difference between RH and indolent NHL (P>0.05). The expression of survivin had a significantly positive correlation with the expression of Ki-67 and FVIIIRAg (r=0.6495, 0.6635, respectively, both P<0.01), and a negative correlation with the expression of caspase3 and AI (r=-0.5820, -0.6013, respectively, P<0.01). It was suggested that survivin may contribute to the progression of NHL by playing an important role in promoting cell proliferation, inhibiting cell apoptosis and enlisting angiogenesis. Survivin expression is closely related to malignant grade and therefore may be considered an important prognostic factor of NHL.