1. Generating Micro- and Nanoliter Flow Rates With a Precolumn Stream Splitter
HPLC pumps function more reproducibly at flow rates above, for example, 10 μL/min, providing typical solvent flow rates in the 0.1-100 μL/min range. These pumps are configured with a static micro-mixer and a variable micro-splitter to produce the specific flow rate required for a given micro-column. Thus, highly reproducible gradients can be achieved with virtually no time delay. For example, a separation of approximately 1:1000 is better than a separation of 1:100. For the latter, pump adjustment is less effective for fine-tuning the flow rate.
1) To obtain a flow rate in the range of 0.1–100 μL/min, connect the T-pieces with capillary tubing of different lengths and internal diameters to give the required percent of the total flow into each line.
2) Test the actual flow rate obtained at a fixed time by connecting the outlet of the HPLC column to a disposable micropipette and note the time taken to fill the micropipette with a stopwatch.
3) Inject a known volume of test solution, such as MRFA that is not retained on the column by the chromatography. Alternatively, using a high organic phase, inject a compound that is not retained under these conditions, such as a peptide, and begin data acquisition. Measure the time to acquire the signal caused by the test compound on the TIC plot. Divide the total time of observation by the volume of the sample.
4) If necessary, adjust the flow rate by cutting an appropriate length from tubing. Typical flow rates are 4 μL/min to 180 nL/min for 1.0 mm, 300 μm, and 75 μm i.d. columns, respectively.
5) A postcolumn stream splitter can be set up in a similar manner to collect part of the eluate from the column and to analyze the rest directly in an electrospray MS.
2 Micro-Column Switching for 2-D Chromatography
A dual gradient pump system includes a capillary LC pump for the first dimensional separation and a nano-LC pump for the second dimensional separation. A column switching module equipped with a miniature 10-port valve and loading pump is used. In addition to gaining additional chromatographic separation dimensions, it allows fast sample loading from a relatively large volume, thus speeding up the analysis. An ion exchange or similar chromatographic step always precedes the reversed-phase step, mainly for compatibility with nanoelectrospray mass spectrometry of solvents eluting peptides from the reversed-phase column.
1) Reduce and alkylate the sample (1 to 400 mg of total protein) with iodoacetamide.
2) Digest the sample with trypsin for 24 h at 37°C.
3) Dilute digested sample with 0.05% TFA to stop the reaction and store at –20°C until required for analysis.
4) Pump sample onto a strong cation-exchange (SCX) column as a first-dimension separation.
5) Apply a gradient at 5–10 μL/min to the SCX column.
6) Elute fractions containing peptides from the column by applying a linear salt gradient. Typical ion-exchange gradient conditions are: start with 0% B (100% buffer A) followed by a 5% increase in buffer B for each fraction over 65 min.
7) Using column switching, subsequently separate the component peptides in each fraction by an acetonitrile/water/formic acid gradient on the reversed-phase column (the second orthogonal separation dimension).
8) Typical reverse-phase gradient conditions are as follows. Mobile phase A comprises 0.1% FA in water. Mobile phase B is 0.08% FA in water/ACN (20:80, v/v%). The gradient is 0% B to 40% B in 80 min, then up to 90% B over 10 min and hold at that percentage for 5 min to wash the column before re-equilibration. Flow rate is 100–300 nL/min.
9) Record the peptide peaks in the UV detector at 214 nm.
10) Analyze the eluant online through a nanospray source.
11) All of each peptide peak may be analyzed or the eluant from the micro-reverse-phase column may be stream split to allow part to be analyzed by electrospray MS (typically 20%) and the rest to be fraction collected or spotted directly onto a MALDI plate using a suitable robot.
3 Peptide Identification by Continuous Nanospray MS Analysis; Use of Self-Pack and Prepacked Fused Silica Columns; Multidimensional Protein Identification Technology (MudPIT)
In these techniques, a single microcapillary column can be loaded with one or two independent chromatographic phases. Since the capillary column is directly attached to the mass spectrometer, no additional sample processing is required once a complex peptide mixture is loaded. The peptides are eluted directly from the column into the tandem mass spectrometer due to the kilovoltage potential directly docked to the microcapillary column. Instead of using two separate columns and multiple switching valves, a dual-phase microcapillary column with sequential strong cation exchange and RP particles is used.
3.1 Prepacked Fused Silica Columns
These are usually prepacked with reverse-phase media for compatibility of eluting solvent with electrospray MS—e.g., PicoFrit nanobore column, 75 mm i.d., with a 5-cm packed bed of C18 5-μm particle size material with a 15-mm integral PicoTip emitter.
1) Identify the nontapered (distal) end of the column.
2) Connect the distal end to the suction pump and adjust the column with 50:50 organic solvent: water solvent according to the manufacturer's instructions.
3) Cut column to required length by making a light score on surface to "nick" the tubing with a diamond-edged scribe.
4) Snap end off and examine at a magnification of ×10–30 to ensure cut is square and smooth. Practice first with fused silica tubing.
5) Sample is loaded onto this micro-column off-line by a micro-flow LC pump.
6) Mount column in flowing/dynamic nanospray head and set up parameters to provide stable electrospray at flow rates of 100–500 nL/min.
7) Occasionally, trim the head of the column to remove accumulated insoluble material if necessary, for prolonged column use.
3.2 Use of Self-Packed Fused Silica Columns
1) Pack a polyimide-coated fused-silica microcapillary column (e.g., 360 o.d. and 75–100 μm i.d., tapered at one end) with 10 cm of 5 μm C18 RP material followed by 4 cm of 5 μm particle strong cation exchange material (SCX).
2) Load up to 400 μg of digested protein sample into the microcapillary column, using a microbore pump.
3) Couple the column to the ion-trap mass spectrometer equipped with a nanoLC electrospray ionization source.
4) Set up an automated 15-step chromatography run as follows, to first displace the peptides from the SCX to the RP by the salt gradient, then elute these off the RP column into the MS/MS.
5) Re-equilibrate the microcolumn and apply an additional salt step of higher concentration to displace more peptides from the SCX to the RP.
6) Elute these peptides with the RP gradient into the mass spectrometer.
4 Peak Parking for NanoLC/Nanospray/MS/MS
Peak parking is the ability to rapidly reduce the gradient flow rate. This provides the mass spectrometer with more time to analyze a specific peak. For example, it may take 10 to 15 seconds to complete an MS/MS of a peptide. If a peak is 30 seconds wide, the mass spectrometer will only be able to analyze the three most abundant peptides and the less abundant peptides will be lost. With peak parking, the flow rate can be slowed by a factor of 10 or more, allowing all peptides in the peak to be analyzed. This allows for a significant increase in the acquisition time of the nanospray mass spectrometry in order to interrogate a larger number of precursor ions without affecting the chromatographic separation.
The system requires the MS to send a signal (contact closure) at the beginning and end of peak identification, or when the instrument is switched from MS to MS/MS mode. Immediately after the initial fraction of the fraction (signaled by the peak) enters the mass spectrum, the flow rate is reduced (e.g., by a factor of 8, from 200 nL to 25 nL/min), which results in a significant increase in interrogation time for the remaining fraction of the peak. Therefore, enhanced precursor ion selection as well as increased MS/MS acquisition times are possible without loss of separation performance or mass spectrometry sensitivity.
5 Robotic System for Fractionation, Spotting, and Preparation
Fractions are collected online from capillary/nanoLC or CZE systems using a robot in volumes as small as a few nanoliters. Collection can be performed on many different types of substrates, such as MALDI-TOF/MS targets; polyvinylidene fluoride (PVDF) membranes for subsequent protein sequencing; 96, 384, or 1536 well plates; or any other collection vessel. The most accurate robot moves the target table rather than the needle. The needle remains in a fixed position, allowing precise nanoliter volumes to be nabbed with better than 20 μm accuracy. these robotic instruments can pipette or dispense samples and reagents. The robot can add MALDI matrix or make-up solution with a dose pump while dispensing fractions onto the MALDI target. Substrates are added coaxially at the tip of the needle to produce optimal spot size.
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Reference
- Walker, J. M. (Ed.). (2005). The proteomics protocols handbook. Humana press.
