A Microscale Electrospray Interface for Online, Capillary Liquid Chromatography/Tandem Mass Spectrometry of Complex Peptide Mixtures

Abstract

A microcapillary liquid chromatography (HPLC) system designed for the gradient elution of peptide and protein samples at flow rates < 1 microL/min has been coupled to a triple-sector quadrupole mass spectrometer via a simple sheathless electrospray interface (microspray). The microspray interface used a flame-drawn, uncoated, fused silica needle with tip outer diameters in the range of 15-20 microm and an opening less than 5 microm in diameter. Online sample filtration to prevent clogging of the drawn needle was accomplished by using a hydrophilic PVDF membrane filter integrated into the needle assembly. The spray potential (0.5-1 kV) was applied directly to the sample stream through the capillary union. Stable electrospray conditions were obtained over the full range of the gradient (0-90% acetonitrile in water) and was generally independent of flow rate. Both off-line and online analyses of proteins and peptide digest mixtures were performed at sample levels less than 10 fmol. HPLC parameters could be optimized for either rapid LC/MS analysis or enhanced performance in LC/MS/MS experiments by modulation of the eluting peak widths. Additionally, flow could be greatly reduced as selected components pass through the interface to prolong the time available to collect mass spectral data. The reduced spectral background and peak width manipulation facilitated the acquisition of peptide production spectra (MS/MS) using real-time, automated instrument control procedures.

A practical guide to nano‐LC troubleshooting

“…Other improvements made by Davis et al [6] and Gatlin et al [7] were based on the integration of column packing with the emitter tip, which resulted in a system, free from the after-column void volumes. Reduction of the void volumes to an absolute minimum plays an extremely important role in micro-and nanoscale LC.…”

Stable gradient nanoflow LC-MS

“…An alternate approach involves modification of the LC system to accommodate separations within the nanoflow regime [6,7]. Nanoflow LC-MS offers benefits in terms of sensitivity, solvent consumption, and sample consumption when compared with higher flow rate LC-MS, however, it can be much more difficult to operate.…”

“…Nanoflow LC-MS offers benefits in terms of sensitivity, solvent consumption, and sample consumption when compared with higher flow rate LC-MS, however, it can be much more difficult to operate. Nanoflow sprayers are typically fabricated from tapered fused silica capillary resulting in the need for some type of surface coating [8,9], inserted electrode [10,11], conductive union [6,12,13], or dialysis membrane [14] for application of the electrospray potential. Alternatively, sprayers can be fabricated from conductive materials, such as stainless steel, [15], however, tapered stainless steel sprayers are much more difficult to fabricate than fused silica sprayers.…”

“…Traditional LC systems operated at solvent flow rates that were orders of magnitude higher (hundreds of L/min to mL/min) than those used for mass spectrometry (tens of L/ min), resulting in the need for solvent flow splitters [2]. Some of the subsequent improvements were focused on the use of nebulizer gases [3] and heating [4,5] for stabilization of the electrospray process to accommodate higher LC solvent flows.An alternate approach involves modification of the LC system to accommodate separations within the nanoflow regime [6,7]. Nanoflow LC-MS offers benefits in terms of sensitivity, solvent consumption, and sample consumption when compared with higher flow rate LC-MS, however, it can be much more difficult to operate.…”

A novel Interface for variable flow nanoscale LC/MS/MS for improved proteome coverage

“…The third method for modulation of the rate of delivery of peptides to the mass spectrometer using variable flow was developed by Davis and Lee [14,19]. Their system varied the flow rate of the mobile phase eluting from the reverse phase nanoscale LC column by varying the mobile phase pressure at the head of the column.…”