U.S. Department of Energy

Pacific Northwest National Laboratory

Methods and Instrument Development

1D LC Separations

High resolution separations are crucial in working with the complex samples seen in pan-omics research. Our typical separations are performed using 50-75um i.d. columns packed in-house with 3um particles to 40-70cm long and then operated at flow rates ranging from 100-300nL/min. However, we also have extensive experience with smaller particles, smaller column i.d., and lower flow rates but these parameters offer the highest level of robustness and reproducibility when sample is not limited. We also employ online trapping whenever possible, which is in most cases. This allows for larger volume samples (5-100+uL) to be trapped at much higher flow rates (2-20+uL/min) to reduce instrument duty cycle and to provide efficient de-salting and sample focusing prior to directing the trapped sample onto the analytical column.

2D LC separations

A widely used strategy to reduce sample complexity in peptide and protein work, aimed at mitigating undersampling issues due to inherent MS duty cycle limitations, is to perform two LC separations that are orthogonal to one another. Offline first dimension separations typically involve either ion exchange or high pH reversed phase with concatenation, followed by online LC-MS analysis of the resulting fractions by low pH reversed phase chromatography. Both of these approaches are widely used in our labs, particularly in studies where isobaric labeling is used to minimize interference in recorder ions. However, there are times where sample limitations exclude the opportunity to perform an offline first dimension separation. In this case, we have developed a robust, fully automated 2D online LC system and routinely use it where sample quantities are limited.

Multi-Column LC systems

Our lab has used multi-column LC systems for over a decade with the aim of maximizing throughput. With a multi-column LC system, the steps in a typical separation that do not result in any useful data are all done offline (sample loading, column dead volume time, column re-equilibrations). An added feature of multi-column LC systems is that extensive off-line column washing routines can be performed that would normally not be possible without greatly extending the time between runs.

Chemically etched emitters

Developed in our lab, chemically etched emitters have played a significant role in improving the robustness of our LC-MS platforms. The internal diameter is easily controlled, because it is unchanged from whatever the initial fused silica i.d. is selected for etching. We use 20um i.d. etched emitters for most applications but prepare these in-house and often use other diameters depending on the needs of the particular project or study.

Specialty LC applications

We have eight LC systems that are custom built and controlled by PNNL software LCMSNet. These custom LC systems were designed for rapid and radical reconfiguration so that we can quickly implement virtually any special instrument configuration for testing. Up to eight 2-position valves and up to 4 multi-position valves are readily available on these systems and pieces of equipment can readily be added as plug-ins (autosamplers, pumps, additional valves, etc). We routinely use these LC systems for various multi-column and 2D configurations but have also used them for many other applications as well such as; online immobilized trypsin digestion, online microscale depletion, high-volume high throughput infusion, and online enrichment/collection.

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