Over the past decade, researchers have approached efficient, cost-effective, mass spectrometry (MS)-based deep proteome analysis as a primary goal. Using yeast as the preferred subject, various research teams have progressed the body of proteomic knowledge. For purposes of standardization, comprehensive analysis refers to the identification of >/= 4,000 proteins, or approximately 90% of the yeast proteome.
Unfortunately, attempts to characterize the proteome face restraints of time, resolution capability and cost. One of the earliest full-scale studies used two-dimensional chromatography coupled with tandem mass spectrometry (MS/MS) to identify 1,483 proteins in approximately 68 hours, or 0.4 proteins per minute.1 With the introduction of hybrid technology, researchers achieved the identification of 2,003 proteins in 48 hours, or 0.7 proteins per minute.2 Orbitrap instrumentation with extensive fractionation allowed for the first comprehensive analysis, resulting in the identification of nearly 4,000 proteins in approximately 144 hours, or 0.5 proteins per minute.3 More recently, researchers focused on increasing quality parameters for both sample preparation and online separations and used streamlined quadrupole-Orbitrap without fractionation to identify over 3,900 proteins in 4 hours, or 16.3 proteins per minute.4 Previous studies have also examined time-of-flight (TOF) instrumentation but found that the spectral quality was poor.5
Recently, Hebert et al.6 presented evidence that a rapid yet accurate analysis of the deep proteome is feasible. They achieved an astounding 67 proteins per minute and completed a comprehensive proteome analysis (4,002 proteins) in just over one hour. To do this, they used a new generation hybrid MS machine (Orbitrap Fusion) with a mass filter, collision cell, Orbitrap analyzer, and dual-cell linear ion trap analyzer (Thermo Scientific) that ensures high acquisition speeds (20 Hz). The researchers combined this technology with careful sample preparation, including an extended bead beating method for cellular lysis, trypsin digestion, and the addition of dimethyl sulfoxide for increased abundance and charge unification.
Hebert et al. point to the use of high mass accuracy and resolution as well as considerable enhancements in MS/MS acquisition speeds to explain their results. In terms of specific experimental parameters, they found that MS/MS analysis with high energy collision dissociation (HCD) and ion trap mass analysis produced greater identifications, particularly when set to a resolving power of 60,000 (m/z 200) with a 35-msec optimal maximum injection time and 0.9 m/z isolation width for the first MS run. Dynamic exclusion settings of 30, 45, and 60 seconds each produced similar results. Ideal automatic gain control target values for the two MS runs were 500,000 and 7,000, respectively. The researchers preferred 30-cm capillary liquid chromatography columns loaded with 1.7-um BEH particles, with 350–375 nL/minute flow rates maintained at a constant temperature on a one-hour gradient.
The researchers demonstrated that the Orbitrap Fusion identifies approximately 8 peptides per second and, at time, achieves up to 19 peptides per second, thereby doubling the efficiency of previous instrumentation. Interestingly, the evaluated machine also offered increased depth of identification, with a mean precursor depth equivalent to the 349th most abundant peak and frequent sampling out to the 800th most abundant peak. Although the researchers expected that increased speed played the largest role in the advancement of comprehensive proteome analysis, they expressed surprise that shorter analysis times also brought increased sensitivity. They suggest that the option to coelute without excessive ionization suppression, coupled with a continued trajectory of rate doubling (from 20 to 40 Hz over the next few years), could allow researchers to comprehensively characterize the human proteome within a one- to two-hour time frame. This possibility has clinical significance, since it would minimize reliance on the transcriptome, which offers little insight into post-translational regulation.
1. Washburn, M.P., et al. (2001) “Large-scale analysis of the yeast proteome by multidimensional protein identification technology,” Nature Biotechnology, 19 (pp. 242–47).
2. de Godoy, L.M.F., et al. (2006) “Status of complete proteome analysis by mass spectrometry: SILAC labeled yeast as a model system,” Genome Biology, 7.
3. de Godoy, L.M.F., et al. (2008) “Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast,” Nature, 455 (pp. 1251–54).
4. Nagaraj, N., et al. (2012) “System-wide Perturbation Analysis with Nearly Complete Coverage of the Yeast Proteome by Single-shot Ultra HPLC Runs on a Bench Top Orbitrap,” Molecular & Cellular Proteomics, 11.
5. Andrews, G.L., et al. (2011) “Performance Characteristics of a New Hybrid Quadrupole Time-of-Flight Tandem Mass Spectrometer (TripleTOF 5600),” Analytical Chemistry, 83 (pp. 5442–46).
6. Hebert, A., et al. (2013, October 19) “The One Hour Yeast Proteome,” Molecular & Cellular Proteomics, Manuscript M113.034769.
Post Author: Melissa J. Mayer. Melissa is a freelance writer who specializes in science journalism. She possesses passion for and experience in the fields of proteomics, cellular/molecular biology, microbiology, biochemistry, and immunology. Melissa is also bilingual (Spanish) and holds a teaching certificate with a biology endorsement.