Spatial Proteomics toward Subcellular Resolution by Coupling Deep Ultraviolet Laser Ablation with Nanodroplet Sample Preparation.

Multiplexed molecular profiling of tissue microenvironments, or spatial omics, can provide critical insights into cellular functions and disease pathology. The coupling of laser microdissection with mass spectrometry-based proteomics has enabled deep and unbiased mapping of >1000 proteins. However, the throughput of laser microdissection is often limited due to tedious two-step procedures, sequential laser cutting, and sample collection. The two-step procedure also hinders the further improvement of spatial resolution to <10 µm as needed for subcellular proteomics. Herein, we developed a high-throughput and high-resolution spatial proteomics platform by seamlessly coupling deep ultraviolet (DUV) laser ablation (LA) with nanoPOTS (Nanodroplet Processing in One pot for Trace Samples)-based sample preparation. We demonstrated the DUV-LA system can quickly isolate and collect tissue samples at a throughput of ∼30 spots/min and a spatial resolution down to 2 µm from a 10 µm thick human pancreas tissue section. To improve sample recovery, we developed a proximity aerosol collection approach by placing DMSO droplets close to LA spots. We demonstrated the DUV-LA-nanoPOTS platform can detect an average of 1312, 1533, and 1966 proteins from ablation spots with diameters of 7, 13, and 19 µm, respectively. In a proof-of-concept study, we isolated and profiled two distinct subcellular regions of the pancreas tissue revealed by hematoxylin and eosin (H&E) staining. Quantitative proteomics revealed proteins specifically enriched to subcellular compartments.

Liquid Chromatographic Separation Using a 2 µm i.d. Open Tubular Column at Elevated Temperature.

We have experimentally demonstrated the extraordinarily high resolving power of liquid chromatography (LC) using a narrow open tubular (OT) column. In this work, we show that we can further increase its efficiency, peak capacity, and separation speed by elevating the operation (or column) temperature; all of these three numbers can be improved without mutual compromises. We use a mixture of five amino acids as a sample and show that we can increase the efficiency by 34%-260% and the separation speeds by 7%-10% by raising the operation temperature from 30 to 70 °C. When we use a 2 µm i.d. × 80 cm in length OT column coated with OTMS at a temperature of 70 °C, we can frequently obtain peak capacities of 700-800 within 20-30 min for separating cytochrome C digests. By increasing the column length to 160 cm, we can obtain a peak capacity of 2720 within 143 min for separating a complex peptide sample. This peak capacity is the highest peak capacity to date for one-dimensional LC separations. Importantly, heating the column is easy to implement and does not cost much, and many commercial LC systems already have compartments to control column temperatures. Running LC using a narrow OT column at an elevated temperature should broaden the applications of OT-LC in chemical and biochemical analyses.

Performing flow injection chromatography using a narrow open tubular column.

Flow injection chromatography (FIC) or sequential injection chromatography (SIC) is a low-pressure liquid chromatography technique that uses flow injection or sequential injection hardware. Due to the constraints of this hardware, the separation resolution is low; often no more than 3-5 components are resolved. We have recently demonstrated the excellent resolving power of narrow open tubular (OT) columns for various biomolecules, and only moderate elution pressures are needed to carry out these separations. In this paper, we incorporate a narrow OT column with FIC and construct an FIC system using a pressure chamber and two injection valves to implement gradient elution. The resultant system not only improves the resolution but also reduces the system cost. When we use the system to separate peptides from trypsin-digested cytochrome C, we can resolve dozens of peptides (with resolutions of 0.5 or greater) at a speed of 12 samples per hour. When we use this system to separate a mixture containing 3 amino acids, we can base-line resolve these compounds at a speed of 1800 sample per hour.

Picoflow Liquid Chromatography-Mass Spectrometry for Ultrasensitive Bottom-Up Proteomics Using 2-µm-i.d. Open Tubular Columns.

In many areas of application, key objectives of chemical separation and analysis are to minimize the sample quantity while maximizing the chemical information obtained. Increasing measurement sensitivity is especially critical for proteomics research, especially when processing trace samples and where multiple measurements are desired. A rich collection of technologies has been developed, but the resulting sensitivity remains insufficient for achieving in-depth coverage of proteomic samples as small as single cells. Here, we combine picoliter-scale liquid chromatography (picoLC) with mass spectrometry (MS) to address this issue. The picoLC employs a 2-µm-i.d. open tubular column to reduce the sample input needed to greatly increase the sensitivity achieved using electrospray ionization (ESI) with MS. With this picoLC-MS system, we show that we can identify ∼1000 proteins reliably using only 75 pg of tryptic peptides, representing a 10-100-fold sensitivity improvement compared with the state-of-the-art liquid chromatography (LC) or capillary electrophoresis (CE)-MS methods. PicoLC-MS extends the limit of separation science and is expected to be a powerful tool for single cell proteomics.

Experimentally Validating Open Tubular Liquid Chromatography for a Peak Capacity of 2000 in 3 h.

The advancements in life science research mandate effective tools capable of analyzing large numbers of samples with low quantities and high complexities. As an essential analytical tool for this research, liquid chromatography (LC) encounters an ever-increasing demand for enhanced resolving power, accelerated analysis speed, and reduced limit of detection. Although theoretical studies have indicated that open tubular (OT) columns can produce superior resolving power under comparable elution pressures and analysis times, ultrahigh-resolution and ultrahigh-speed open tubular liquid chromatography (OTLC) separations have never been reported. Here we present experimental results to demonstrate the predicted potential of this technique. We use a 2 µm i.d. × 75 cm long OT column coated with trimethoxy(octadecyl)silane for separating pepsin/trypsin digested E. coli lysates and routinely produce exceptionally high peak capacities (e.g., 1900-2000 in 3-5 h). We reduce the column length to 2.7 cm and exhibit the capability of OTLC for ultrafast separations. Under an elution pressure of 227.5 bar, we complete the separation of six amino acids in ∼800 ms and resolve these compounds within ∼400 ms. In addition, we show that OTLC has low attomole limits of detection (LOD) and each separation requires samples of only a few picoliters. Importantly, no ultrahigh elution pressures are required. With the ultrahigh resolution, ultrahigh speed, low LOD, and low sample volume requirement, OTLC can potentially be a powerful tool for biotech research, especially single cell analysis.

Ultrafast Gradient Separation with Narrow Open Tubular Liquid Chromatography.

Separation speed and resolution are two important figures of merit in chromatography. Often, one gains the speed at the cost of the resolution, and vice versa. Scientists have employed short-packed columns for ultrafast separations but encountered challenges such as limited mobile phase velocity, extra-column effect caused band broadening, and column packing difficulty. We have recently demonstrated ultrahigh resolutions of narrow open tubular liquid chromatography (NOTLC); this allows us to trade some of the resolution for speed. In this work, we explored NOTLC for ultrafast LC separations. We used a 2.7 cm (effective length) narrow open tubular (NOT) column and showed a baseline separation of 6 amino acids in less than 700 ms. Ways to further increase the speed were discussed. Using short narrow open tubular (NOT) columns to perform ultrafast separation we overcame the challenges from using short packed columns. To demonstrate the feasibility of using this ultrafast separation technique for practical applications, we separated complex protein digests; peptides were nicely resolved in ∼1 min.

On-column and gradient focusing-induced high-resolution separation in narrow open tubular liquid chromatography and a simple and economic approach for pico-gradient separation.

We have recently obtained extraordinarily high efficiencies and sharp peaks using narrow open tubular (OT) columns for liquid chromatographic separations. On-column focusing is commonly observed in liquid chromatography, but this effect alone could not satisfactorily explain the sharpness of these peaks. In this work we investigated the reasons that could have led to the peak sharpness. We hypothesized initially that analytes confined in a narrow OT column might have significantly reduced analyte diffusivities and the reduced diffusivities consequently resulted in the peak sharpness. This hypothesis was invalidated immediately after we measured the diffusion coefficients and did not notice any noticeable diffusivity increases of the analytes inside such columns. We then designed an experiment and revealed a "re-focusing effect". Investigation of this re-focusing effect eventually led us to the observation of a gradient focusing caused by the composition difference between the eluent and the sample matrix. It was this gradient focusing that had contributed primarily to the peak sharpness. On the basis of this insightful understanding, we further developed a simple and economic approach to perform pico-gradient narrow open tubular liquid chromatographic separations.

Narrow, Open, Tubular Column for Ultrahigh-Efficiency Liquid-Chromatographic Separation under Elution Pressure of Less than 50 bar.

We report that we can achieve extremely high separation efficiencies using a narrow, open, tubular (NOT) column for liquid-chromatographic separations, and we can carry out these separations under an elution pressure of no more than 50 bar. To improve the separation efficiency in packed-column liquid chromatography, one of the most effective approaches is to reduce the monodispersed-particle sizes. A direct consequence of reduced particle size is an increased elution pressure. High efficiencies have been obtained in ultrahigh-performance liquid chromatography (UPLC) using 1-2 µm or even submicron particles, and high elution pressures (greater than 1000 bar) are commonly used to carry out these separations. Open, tubular (OT) columns have been predicted to be the most efficient columns for high-efficiency liquid-chromatographic separations, as long as the column diameter is sufficiently small (1-2 µm). However, high efficiencies have not yet been publically reported, possibly because of the challenges (such as picoliter-volume detection, nanocapillary-column preparation, low sample loadability, etc.) of utilizing 1-2 µm diameter capillaries. In this paper, we show how we overcame these problems and achieved extremely high separation efficiencies using a 2 µm inner diameter capillary. We see 200+ apparent peaks with a peak capacity of 810 within 54 min when separating a sample from trypsin-digested cytochrome C, and we count 440 apparent peaks with a peak capacity of 1640 within 172 min when separating a sample from pepsin/trypsin-digested Escherichia coli cell lysate.

A narrow open tubular column for high efficiency liquid chromatographic separation.

We report a great feature of open tubular liquid chromatography when it is run using an extremely narrow (e.g., 2 µm inner diameter) open tubular column: more than 10 million plates per meter can be achieved in less than 10 min and under an elution pressure of ca. 20 bar. The column is coated with octadecylsilane and both isocratic and gradient separations are performed. We reveal a focusing effect that may be used to interpret the efficiency enhancement. We also demonstrate the feasibility of using this technique for separating complex peptide samples. This high-resolution and fast separation technique is promising and can lead to a powerful tool for trace sample analysis.