Ultralow-Volume Fraction Collection from NanoLC Columns for Mass Spectrometric Analysis of Protein Phosphorylation and Glycosylation

Abstract

An ultralow volume fraction collection system referred to as nano fraction analysis chip technology (nanoFACT) is reported. The system collects 25-2500-nL fractions from 75-microm nanoLC columns into pipet tips at a user-defined, timed interval, typically one fraction every 15-120 s. Following collection, the fractions in the tip dry down naturally on their own in such a way as to create a concentrated band at the very end of the interior of the pipet tip. The fractions are then reconstituted directly in the pipet tips in approximately 250 nL of solvent prior to analysis. Because the chromatography and reconstitution solvent are independent, the reconstitution solvent can be selected to maximize ionization efficiency without compromising chromatography. In the infusion analysis of the nanoLC fractions, a low-flow electrospray chip is used which consists of 400 nozzles, each with an inner diameter of 2.5 microm and yielding flow rates of approximately 20 nL/min. Therefore, when reconstituted in 250 nL, each nanoLC fraction can be analyzed for over 10 min. This increase in analysis time allows for signal averaging, resulting in higher data quality, collision energy optimization, slower scanning techniques to be used, such as neutral loss and precursor ion scanning, higher resolution scans on FTMS instruments, and improved peptide quantitation. Furthermore, the nanoLC fractions could be archived in the pipet tips for analysis at a later date. Here, the advantages of nanoFACT are shown for phosphorylation analysis using bovine fetuin and glycosylation analysis using bovine ribonuclease B (RNase B). In the phosphorylation analysis, a comparison between conventional nanoLC and a nanoFACT analysis was performed. An MS/MS spectrum of a triply phosphorylated peptide, 313-HTFSGVApSVEpSpSSGEAFHVGK-333 could only be obtained using nanoFACT, not with nanoLC. Furthermore, spectral quality for the nanoFACT analysis was significantly improved over nanoLC. This was determined by comparing the number of diagnostic ions between the nanoFACT and nanoLC spectra, and it was found that the nanoFACT spectra contained a 19% or greater number of diagnostic ions for nonphosphorylated peptides and 55% or greater for phosphorylated peptides. For the glycosylation analysis, the glycosylation site of RNase B was fully characterized using 100 fmol of tryptic digest on a three-dimensional ion trap mass spectrometer.