Nano-LC in Proteomics: Recent Advances and Approaches

Wilson, Steven Ray; Vehus, Tore; Berg, Henriette Sjaanes; Lundanes, Elsa

doi:10.4155/bio.15.92

Cited by 129 publications

(89 citation statements)

References 114 publications

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“…Due to reduced volumetric flow rates and increased ionization efficiency, cLC couples particularly well with ESI-MS. 1–4 This has led to cLC’s dominance in the fields of proteomics, 5–10 metabolomics, 3,7,11–16 and in vivo determination of neuropeptides. 17–21 Combined with gradient elution, peak capacities exceeding 800 22,23 have been achieved.…”

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confidence: 99%

Improving the Sensitivity, Resolution, and Peak Capacity of Gradient Elution in Capillary Liquid Chromatography with Large-Volume Injections by Using Temperature-Assisted On-Column Solute Focusing

2016

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Capillary HPLC (cLC) with gradient elution is the separation method of choice for the fields of proteomics and metabolomics. This is due to the complementary nature of cLC flow rates and electrospray or nanospray ionization mass spectrometry (ESI-MS). The small column diameters result in good mass sensitivity. Good concentration sensitivity is also possible by injection of relatively large volumes of solution and relying on solvent-based solute focusing. However, if the injection volume is too large or solutes are poorly retained during injection, volume overload occurs which leads to altered peak shapes, decreased sensitivity, and lower peak capacity. Solutes that elute early even with the use of a solvent gradient are especially vulnerable to this problem. In this paper, we describe a simple, automated instrumental method, temperature-assisted on-column solute focusing (TASF), that is capable of focusing large volume injections of small molecules and peptides under gradient conditions. By injecting a large sample volume while cooling a short segment of the column inlet at subambient temperatures, solutes are concentrated into narrow bands at the head of the column. Rapidly raising the temperature of this segment of the column leads to separations with less peak broadening in comparison to solvent focusing alone. For large volume injections of both mixtures of small molecules and a bovine serum albumin tryptic digest, TASF improved the peak shape and resolution in chromatograms. TASF showed the most dramatic improvements with shallow gradients, which is particularly useful for biological applications. Results demonstrate the ability of TASF with gradient elution to improve the sensitivity, resolution, and peak capacity of volume overloaded samples beyond gradient compression alone. Additionally, we have developed and validated a double extrapolation method for predicting retention factors at extremes of temperature and mobile phase composition. Using this method, the effects of TASF can be predicted, allowing determination of the usefulness of this technique for a particular application.

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confidence: 99%

Improving the Sensitivity, Resolution, and Peak Capacity of Gradient Elution in Capillary Liquid Chromatography with Large-Volume Injections by Using Temperature-Assisted On-Column Solute Focusing

2016

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“…3). Tryptic peptides were equally or better resolved on the attoLC-MS system compared to the standard system, with a peak capacity (n c ** )19 up to 250 at 10 nL/min, compared to 130 at 200 nL/min (same t G /t M , gradient time/system hold-up time) (Supplementary Figure 6A). …”

Section: Resultsmentioning

confidence: 99%

“…Central challenges in miniaturized separation systems include low sample capacity19, under-par column-to-column performance19 and reduced chromatographic robustness. We have developed an OTLC-MS system (attoLC-MS) with repeatable manufacturing of columns and set-up.…”

Section: Discussionmentioning

confidence: 99%

Versatile, sensitive liquid chromatography mass spectrometry – Implementation of 10 μm OT columns suitable for small molecules, peptides and proteins

Vehus

Røberg-Larsen

Waaler

et al. 2016

Sci Rep

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We have designed a versatile and sensitive liquid chromatographic (LC) system, featuring a monolithic trap column and a very narrow (10 μm ID) fused silica open tubular liquid chromatography (OTLC) separation column functionalized with C18-groups, for separating a wide range of molecules (from small metabolites to intact proteins). Compared to today’s capillary/nanoLC approaches, our system provides significantly enhanced sensitivity (up to several orders) with matching or improved separation efficiency, and highly repeatable chromatographic performance. The chemical properties of the trap column and the analytical column were fine-tuned to obtain practical sample loading capacities (above 2 μg), an earlier bottleneck of OTLC. Using the OTLC system (combined with Orbitrap mass spectrometry), we could perform targeted metabolomics of sub-μg amounts of exosomes with 25 attogram detection limit of a breast cancer-related hydroxylated cholesterol. With the same set-up, sensitive bottom-up proteomics (targeted and untargeted) was possible, and high-resolving intact protein analysis. In contrast to state-of-the-art packed columns, our platform performs chromatography with very little dilution and is “fit-for-all”, well suited for comprehensive analysis of limited samples, and has potential as a tool for challenges in diagnostics.

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“…ESI is inherently inefficient at higher flow rates. In the 1990s, multiple groups demonstrated improved ionization efficiency at lower flow rate and developed variants of ESI (ie, microelectrospray, later renamed nanospray) . As analyte concentration also remains highest at low LC flows, capillary LC (typical ID ≤ 100 μm and flow rate ≤1 μL/minute) with nanospray MS soon dominated proteomics research.…”

Section: Case Study: Overcoming Limitations Of Lc‐msmentioning

confidence: 99%

Developing front‐end devices for improved sample preparation in MS‐based proteome analysis

Doucette

Nickerson

2020

J Mass Spectrom

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Chemical analysis has long relied on instrumentation, from the simplest (eg, burets) to the more sophisticated (eg, mass spectrometers) to facilitate precision measurements. Regardless of their complexity, the development of a new instrumental device can be a valued approach to address problems in science. In this perspective, we outline the process of novel device design, from early phase conception to the manufacturing and testing of the tool or gadget. Focus is placed on the development of improved front‐end devices to facilitate protein sample manipulations ahead of mass spectrometry, which therefore augment the proteomics workflow. Highlighted are some of the many training secrets, choices, and challenges that are inherent to the often iterative process of device design. In hopes of inspiring others to pursue instrument design to address relevant research questions, we present a summary list of points to consider prior to innovating their own devices.

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Nano-LC in Proteomics: Recent Advances and Approaches

Cited by 129 publications

References 114 publications

Improving the Sensitivity, Resolution, and Peak Capacity of Gradient Elution in Capillary Liquid Chromatography with Large-Volume Injections by Using Temperature-Assisted On-Column Solute Focusing

Improving the Sensitivity, Resolution, and Peak Capacity of Gradient Elution in Capillary Liquid Chromatography with Large-Volume Injections by Using Temperature-Assisted On-Column Solute Focusing

Versatile, sensitive liquid chromatography mass spectrometry – Implementation of 10 μm OT columns suitable for small molecules, peptides and proteins

Developing front‐end devices for improved sample preparation in MS‐based proteome analysis

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