Fast Liquid Chromatography Separation Research Articles

A rapid and sensitive single-cell proteomic method based on fast liquid-chromatography separation, retention time prediction and MS1-only acquisition

Single-cell analysis has received much attention in recent years for elucidating the widely existing cellular heterogeneity in biological systems. However, the ability to measure the proteome in single cells is still far behind that of transcriptomics due to the lack of sensitive and high-throughput mass spectrometry methods. Herein, we report an integrated strategy termed “SCP-MS1” that combines fast liquid chromatography (LC) separation, deep learning-based retention time (RT) prediction and MS1-only acquisition for rapid and sensitive single-cell proteome analysis. In SCP-MS1, the peptides were identified via four-dimensional MS1 feature (m/z, RT, charge and FAIMS CV) matching, therefore relieving MS acquisition from the time consuming and information losing MS2 step and making this method particularly compatible with fast LC separation. By completely omitting the MS2 step, all the MS analysis time was utilized for MS1 acquisition in SCP-MS1 and therefore led to 65%–138% increased MS1 feature collection. Unlike “match between run” methods that still needed MS2 information for RT alignment, SCP-MS1 used deep learning-based RT prediction to transfer the measured RTs in long gradient bulk analyses to short gradient single cell analyses, which was the key step to enhance both identification scale and matching accuracy. Using this strategy, more than 2000 proteins were obtained from 0.2 ng of peptides with a 14-min active gradient at a false discovery rate (FDR) of 0.8%. Comparing with the DDA method, improved quantitative performance was also observed for SCP-MS1 with approximately 50% decreased median coefficient of variation of quantified proteins. For single-cell analysis, 1715 ± 204 and 1604 ± 224 proteins were quantified in single 293T and HeLa cells, respectively. Finally, SCP-MS1 was applied to single-cell proteome analysis of sorafenib resistant and non-resistant HepG2 cells and revealed clear cellular heterogeneity in the resistant population that may be masked in bulk studies.

HTLC: A Viable Approach to Fast LC of Biogenic Amines in Food with Legacy Equipment

Nowadays, there is a demand for fast liquid chromatography (LC) of biogenic amines in food with the equipment available in the average laboratory. To this end, high-temperature liquid chromatography (HTLC) is presented in this study as a viable option, working on a conventional LC platform at moderately high temperatures up to 80 °C. The LC platform was adapted by including an appropriate length of stainless-steel microbore tubing as a preheater. Biogenic amines as benzoyl derivatives showed good thermostability on a thermally rugged column (150 × 4.6 mm, 5 µm). As a model application the HTLC conditions were developed for wine separation. The separation selectivity changed considerably with temperature in the range 36–84 °C, and baseline resolution was obtained only at temperatures well above ambient. At 73 °C oven temperature, the system allowed a flow rate as high as 3 mL min−1 with the backpressure not exceeding 195 bar (2800 psi). The separation took less than 5 min, comparably fast to documented fast LC separations, and typically at least three to five times faster than a conventional separation.

Fast liquid chromatography/tandem mass spectrometry (highly selective selected reaction monitoring) for the determination of toltrazuril and its metabolites in food

In this work a fast liquid chromatography (LC)-tandem mass spectrometry (MS/MS) method was developed for the analysis of toltrazuril, a coccidiostatic drug, and its metabolites in meat food products. The applicability of atmospheric pressure chemical ionization (APCI) and heated electrospray ionization in both positive and negative modes was studied. APCI in negative mode provided the best results and the base peak originated from the loss of CF(3) (toltrazuril and toltrazuril sulfone) and CHF(3)* (toltrazuril sulfoxide) was used as the precursor ion in MS/MS. A fast LC separation on a C(18) Fused-Core column was used together with the APCI-MS/MS method developed using enhanced mass resolution mode (highly selective selected reaction monitoring, H-SRM) to improve the sensitivity and selectivity for the analysis of these compounds in food samples. A simple sample treatment based on an extraction with acetonitrile and a cleanup with a C(18) cartridge was used. The LC-MS/MS (H-SRM) method showed good precision (relative standard deviation lower than 10%), accuracy, and linearity and allowed the determination of these compounds in food samples down to the parts per billion level (limits of detection between 0.5 and 5 microg kg(-1)).

Fast liquid chromatography separation and multiple‐reaction monitoring mass spectrometric detection of neurotransmitters

We describe here the fast LC-MS/MS separation of a mixture of neurotransmitters consisting of dopamine, epinephrine, norepinephrine, 3,4-dihydroxybenzylamine (DHBA), salsolinol, serotonin, and gamma-aminobutyric acid (GABA). The new UltiMate 3000 Rapid Separation system (RSLC) was successfully coupled to the 4000 QTRAP mass spectrometer operating in multiple-reaction monitoring (MRM) mode. The separation was attained using a 100 mm length, 2.2 microm particle size Acclaim column at a flow rate of 0.5 mL/min. The column back pressure was 350 bar, while the total run time including column re-equilibration was 5.2 min. The peak resolution was minimally affected by the fast separation. The RSLC-MRM separation was found to have a precision range based on peak area for 50 replicate runs of 2-5% CV for all analytes, and the reproducibility of the retention time for all analytes was found to range from 0-2% CV. The described method represents an almost seven times shorter analysis time of neurotransmitters using LC/MRM which is very useful in screening large quantities of biological samples for various neurotransmitters.

Differential 12C-/13C-Isotope Dansylation Labeling and Fast Liquid Chromatography/Mass Spectrometry for Absolute and Relative Quantification of the Metabolome

We report a new quantitative metabolome profiling technique based on differential (12)C-/(13)C-isotope dansylation labeling of metabolites, fast liquid chromatography (LC) separation and electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) detection. An isotope reagent, (13)C-dansyl chloride, can be readily synthesized. This reagent, along with (12)C-dansyl chloride, provides a simple and robust means of labeling metabolites containing primary amine, secondary amine, or phenolic hydroxyl group(s). It is shown that dansylation labeling offers 1-3 orders of magnitude ESI signal enhancement over the underivatized counterparts. Dansylation alters the chromatographic behaviors of polar and ionic metabolites normally not retainable on a reversed phase (RP) column to an extent that they can be retained and separated by RPLC with high efficiency. There is no isotopic effect on RPLC separation of the differential isotope labeled metabolites, and (12)C-/(13)C-labeled isoforms of metabolites are coeluted and detected by MS for precise and accurate quantification and confident metabolite identification. It is demonstrated that, in the analysis of 20 amino acids, a linear response of over 2 orders of magnitude is achieved for relative metabolite quantification with an average relative standard deviation (RSD) of about 5.3% from replicate experiments. A dansylation standard compound library consisting of 121 known amines and phenols has been constructed and is proven to be useful for absolute metabolite quantification and MS-based metabolite identification in biological samples. As an example, the absolute concentrations of 93 metabolites, ranging from 30 nM to 2510 microM, can be determined from a pooled sample of human urines collected in 5 consecutive days labeled with (12)C-dansylation and spiked with the 121 (13)C-dansylated standards. Relative concentration variations of these metabolites in individual urine samples can also be monitored by mixing the (13)C-dansylated pooled urine sample with the (12)C-dansylated individual sample. With a 12 min fast LC separation combined with FTICR MS, 672 metabolites were detected in a human urine sample with each metabolite peak having a signal-to-noise ratio of greater than 20; the identities of most of the metabolites remain to be determined. This work illustrates that dansylation labeling and fast LC/FTICR MS can be a powerful technique for quantitative profiling of at least 672 metabolites in urine samples in 12 min.

Liquid chromatography system for fast, accurate analysis

The design characteristics of a liquid chromatograph that are essential for very fast separations are examined theoretically. Equations are derived that permit the calculation of the column length, column diameter, maximum instrument time constant and flow-rate to effect a given separation in the minimum time. A gradient elution system, that can provide gradients covering a wide range of solvent concentrations and gradient profiles that can be delivered in a few seconds, is disclosed. Examples are given of the rapid separation of multi-component mixtures in less than 30 sec by both isocratic and gradient elution. The separation times obtained experimentally are compared with those predicted theoretically. The quantitative precision that was obtained from fast liquid chromatography separations is also reported.