Enhanced peptide identification with a high-res MS

Analysis of biological samples frequently involves the identification of peptides from low amounts of complex samples. Confident identification of these peptides requires rapid generation of high-quality, high-sensitivity MS and MSMS data.

For complex samples, resolution is required from both the LC and the mass spectrometer. Modern-day LC systems exploit rapid LC gradients which offer increased speed of analysis. Very narrow peak widths are produced, commonly only 1 second wide compared to 30 seconds with a standard LC. This effectively increases the peptide concentration reaching the mass spec and therefore results in increased sensitivity. It is, however, essential that the mass spec duty cycle is fast enough to detect these peaks as they rapidly emerge from the LC system and that enough data points are acquired from each peak profile, particularly if quantitation is desired.

Experimental

Complex sample: 100 ng of E. coli cells. Low abundance sample: 500 amol Bovine serum albumin (BSA). Both samples were digested with trypsin.

LC systems and gradients: Ultimate 3000 (Dionex) and EASY-nLC (Bruker). Gradient: from 5 to 40% ACN, 0.1% formic acid, in 90 min (E. coli) or 10 min (BSA), flow rate 300 nL/min. Analytical column: 75 µm × 150 mm, trapping column, 100 µm × 10 mm; Acclaim PepMap100 C18(Dionex).

MS system: Bruker Daltonics maxis ultrahigh-resolution TOF.

Results

Complex sample: From 100 ng of digested E. coli cells, 690 proteins were identified with a stringent false positive detection rate of 0.57% (Figure 1). 8507 spectra were acquired and searched by Mascot 2.2. The Swissprot 56.1 database was used, with taxonomy restricted to E. coli. The modifications included were carbamidomethyl Cys (fixed) and oxidised Met (variable).

Figure 1: ProteinScape screenshot of Protein list (start and end). 690 proteins were identified from 100 ng digested E. coli cells.

4917 peptides matched the identity threshold with an average Mascot score of 58.8. Thus, over 58% of MSMS spectra were identified as significant - demonstrating the high quality of the MSMS data produced. See Figure 2 as an example.

Figure 2: Example fragment spectrum delivering high sequence coverage.

Low-abundance samples: From 500 amol BSA, 13 unique peptides were identified, representing a sequence coverage of 23% and an overall Mascot scoring of 412 (Figure 3). Here, taxonomy was restricted to mammals. Again, peptides were confidently identified based on high-quality MSMS data.

Figure 3: Fragment spectrum from a 500 amol BSA digest.

Hence, these results prove maXis’s capability to provide high MS resolving power to separate potentially overlapping signals in a complex sample and therefore to allow correct mass assignments. Ultimately, these features directly translate into high identification rates with excellent Mascot scores - even on a time scale that keeps pace with modern ultrafast LC systems.

Conclusion

The key proteomics goals are identifying more peptides with more certainty. The maXis ultrahigh-resolution TOF mass spectrometer brings together novel design technologies to reach these goals. The technological advances include a hexapole collision cell together with an ion cooler, which provide efficient fragmentation of ions from a broad mass range and efficient transfer of fragment ions from the collision cell into orthogonal acceleration. In addition, techniques have been employed to reduce energy distribution in the direction of flight and to ensure a precise starting position for orthogonal deflection.

In combination, these technical developments result in high-quality, information-rich spectra at high resolution (50,000) with incredible mass accuracy (low to sub ppm) at the MS and MSMS level along with fast acquisition speeds. A substantial increase in the number of peptides identified from both complex and low-abundance samples with an increase in significance and certainty for the peptides identified has been demonstrated.

By Laura C Main, Ralf Ketterlinus; Bruker Daltonics