An LC-IMS-MS platform providing increased dynamic range for high-throughput proteomic studies

Abstract

A high-throughput approach and platform using 15 min reversed-phase capillary liquid chromatography (RPLC) separations in conjunction with ion mobility spectrometry-mass spectrometry (IMS-MS) measurements was evaluated for the rapid analysis of complex proteomics samples. To test the separation quality of the short LC gradient, a sample was prepared by spiking 20 reference peptides at varying concentrations from 1 ng/mL to 10 microg/mL into a tryptic digest of mouse blood plasma and analyzed with both a LC-Linear Ion Trap Fourier Transform (FT) MS and LC-IMS-TOF MS. The LC-FT MS detected 13 out of the 20 spiked peptides that had concentrations >or=100 ng/mL. In contrast, the drift time selected mass spectra from the LC-IMS-TOF MS analyses yielded identifications for 19 of the 20 peptides with all spiking levels present. The greater dynamic range of the LC-IMS-TOF MS system could be attributed to two factors. First, the LC-IMS-TOF MS system enabled drift time separation of the low concentration spiked peptides from the high concentration mouse peptide matrix components, reducing signal interference and background, and allowing species to be resolved that would otherwise be obscured by other components. Second, the automatic gain control (AGC) in the linear ion trap of the hybrid FT MS instrument limits the number of ions that are accumulated to reduce space charge effects and achieve high measurement accuracy, but in turn limits the achievable dynamic range compared to the IMS-TOF instrument.

Figure 1

Typical LC-MS base peak chromatograms for the spiked mouse plasma sample with the a) 15.5 and b) 100 minute gradients. An average elution time for each peptide was ~4 seconds with the 15.5-min gradient and ~20 seconds in the 100-min gradient.

Figure 2

The 15.5-min LC-IMS-MS nested spectra summed over all LC elution times for the 20 peptides spiked into a) mobile phase A (standard) and b) mouse plasma.

Figure 3

The 15.5 minute LC-MS spectra of the 10 ng/mL peptides a) (renin)³⁺ and b) (bradykinin fragment 1–7)⁺. The inverted blue spectra from the standard sample show the correct m/z and isotopic data for each peptide, while the red spectra from the mouse plasma sample show the spectra for the elution time when the spiked peptides should be observed.

Figure 4

The 15.5 minute LC-IMS-MS a) summed and b) drift time selected mass spectra for (bradykinin fragment 1–7)⁺. The mouse plasma sample spectra are red and the inverted blue spectra from the standard show the correct isotopic pattern for (bradykinin fragment 1–7)⁺. When all drift times are summed, (bradykinin fragment 1–7)⁺ cannot be observed because a 2+ mouse plasma peptide interferes with its deisotoping, but if drift time selection from 42.7 to 43.7 ms is carried out, bradykinin fragment 1–7 is easily deisotoped.

Figure 5

The nested spectra for the 10 ng/mL peptides a) (renin)³⁺ and b) (bradykinin fragment 1–7)⁺ in the mouse plasma sample acquired with the 15.5 minute LC gradient. The total ion chromatograms (TICs) are displayed above the nested spectra and mass spectra are on the left side. The mass spectra on the left sum all drift times, while the mass spectra on the right only sum a 1 ms drift time window (shown by the red box). By only summing a small window, both (renin)³⁺ and (bradykinin fragment 1–7)⁺ can be deisotoped (deisotoping to the tenths place is shown for clarity).

Figure 6

The nested spectra for 1 ng/mL (fibrinopeptide A)²⁺ acquired with the 15.5 minute LC gradient in mobile phase A for a) and mouse plasma for b) and c). The mass spectrum in b) sums all drift times, while the mass spectrum in c) only sums a 1 ms drift time window (shown in red). Detection and deisotoping of the (fibrinopeptide A)²⁺ is only possible with the drift time window selection.

Figure 7

Average peak area CV values for each spiking level in the mouse plasma sample. The average CV values from all the analyses increase with decreasing concentration. The average LC-IMS-MS peak area CV values were lower than the LC-MS values at all three spiking levels where peptides were detected for all three platforms.