On the scalability and requirements of whole protein mass spectrometry

Abstract

Top-down proteomics has improved over the past decade despite the significant challenges presented by the analysis of large protein ions. Here, the detection of these high mass species by electrospray-based mass spectrometry (MS) is examined from a theoretical perspective to understand the mass-dependent increases in the number of charge states, isotopic peaks, and interfering species present in typical protein mass spectra. Integrating these effects into a quantitative model captures the reduced ability to detect species over 25 kDa with the speed and sensitivity characteristic of proteomics based on <3 kDa peptide ions. The model quantifies the challenge that top-down proteomics faces with respect to current MS instrumentation and projects that depletion of (13)C and (15)N isotopes can improve detection at high mass by only <2-fold at 100 kDa whereas the effect is up to 5-fold at 10 kDa. Further, we find that supercharging electrosprayed proteins to the point of producing <5 charge states at high mass would improve detection by more than 20-fold.

Figure 1

The reduction in S:N for a single isotopic distribution populated by 1 million charges as a function of increasing protein mass. Ions were assumed to have a +1 charge state to eliminate contributions from charge state distributions created by ESI.

Figure 2

The reduction in S:N as a function of increasing mass at various isotopic depletion levels for a single isotopic distribution.

Figure 3

Experimental charge state distributions for proteins ranging from 8.6 – 47 kDa fit by Gaussian distributions.

Figure 4

Decay in S:N as a function of increasing mass resulting from the increasing number of charge states observed for electrosprayed protein ions.

Figure 5

The combined effect of isotopes and charge states on S:N as a function of protein mass.

Figure 6

The effect of isotopic depletion when considered with charge state distributions.

Figure 7

The contribution of various types of chemical noise to the reduction in S:N as a function of precursor mass.

Figure 8

Histogram of protein sizes in the human proteome. Plot was created using 18,852 entries for Homo sapiens using the Uniprot Knowledgebase released on April 4, 2011 and the bin size is 1,000 dalton.