Peptides mass resolution

The m/z values of peptide ions are mathematically derived from the sine wave profile by the performance of a fast Fourier transform operation. Thus, the detection of ions by FTICR is distinct from results from other MS approaches because the peptide ions are detected by their oscillation near the detection plate rather than by collision with a detector. Consequently, masses are resolved only by cyclotron frequency and not in space (sector instruments) or time (TOF analyzers). The magnetic field strength measured in Tesla correlates with the performance properties of FTICR. The instruments are very powerful and provide exquisitely high mass accuracy, mass resolution, and sensitivity—desirable properties in the analysis of complex protein mixtures. FTICR instruments are especially compatible with ESI29 but may also be used with MALDI as an ionization source.30 FTICR requires sophisticated expertise. Nevertheless, this technique is increasingly employed successfully in proteomics studies. 

Fig. 6. Protein identification using a peptide map measured with a matrix-assisted laser desorption time-of-flight mass spectrometer. All the peptide extracted from the gel is measured and the set of masses is used in the database search. The mass resolution is in the order of 10,000. Individual isotopes of a 2.5 kDa peptide are clearly resolved.

He F., Hendrickson C.L., and Marshall A.G. (2001), Baseline mass resolution of peptide isobars a record for molecular mass resolution, Anal. Chem. 73, 647-650. 

He, F. Hendrickson, C.L. Marshall, A.G. Basehne Mass Resolution of Peptide Isobars A Record for Molecular Mass Resolution. Anal Chem. 2001, 73, 647-650. 

In tandem MS mode, because the product ions are recorded with the same TOF mass analyzers as in full scan mode, the same high resolution and mass accuracy is obtained. Isolation of the precursor ion can be performed either at unit mass resolution or at 2-3 m/z units for multiply charged ions. Accurate mass measurements of the elemental composition of product ions greatly facilitate spectra interpretation and the main applications are peptide analysis and metabolite identification using electrospray iomzation [68]. In TOF mass analyzers accurate mass determination can be affected by various parameters such as (i) ion intensities, (ii) room temperature or (iii) detector dead time. Interestingly, the mass spectrum can be recalibrated post-acquisition using the mass of a known ion (lock mass). The lock mass can be a cluster ion in full scan mode or the residual precursor ion in the product ion mode. For LC-MS analysis a dual spray (LockSpray) source has been described, which allows the continuous introduction of a reference analyte into the mass spectrometer for improved accurate mass measurements [69]. The versatile precursor ion scan, another specific feature of the triple quadrupole, is maintained in the QqTOF instrument. However, in pre-... 

Fourier transform mass spectrometry (FTMS) is a rapidly growing technique of increasing analytical importance. Foremost among its many attributes are its high mass resolution and wide mass range capabilities, as well as its ability to store ions. This relatively new technique has been employed in a wide variety of applications, ranging from the exact mass measurement of stable nuclides to the determination of peptide sequences. The future holds considerable promise both for the expanded use of FTMS in a diverse range of chemical problems, as well as advances in the capabilities of the technique itself. 

Laser desorption Fourier transform mass spectrometry (LD-FTMS) results from a series of peptides and polymers are presented. Successful production of molecular ions of peptides with masses up to 2000 amu is demonstrated. The amount of structurally useful fragmentation diminishes rapidly with increasing mass. Preliminary results of laser photodissociation experiments in an attempt to increase the available structural information are also presented. The synthetic biopolymer poly(phenylalanine) is used as a model for higher molecular weight peptides and produces ions approaching m/z 4000. Current instrument resolution limits are demonstrated utilizing a polyethylene-glycol) polymer, with unit mass resolution obtainable to almost 4000 amu. 

A time-of-flight (TOF) analyzer allows positively charged peptides to fly through a vacuum tube toward a negatively charged detector source, so that the time of flight can be calibrated in proportion to peptide mass. Typically, the longer the vacuum tube, the more accurate the mass will be because of increased resolution. 

In general, MALDI of samples fixed to membranes resulted in no loss of mass resolution or mass range. Spectra were extremely reproducible, and could usually be acquired at a lower threshold laser intensity. Figure 1 shows a peptide and protein mixture desorbed from each of the 5 tested membranes. All membranes except the CIS extraction disk produced well resolved spectra. Higher masses were better resolved in samples fixed to polyethylene membranes, while lower masses were better resolved by fixing samples to the Type 61 disposable IR card. Doubly and triply charged ions formed more readily upon desorption/ionization from all the membranes tested, than from stainless steel surfaces. 

Improvement in mass resolution by MALDI of samples loaded on synthetic membranes was particularly apparent in the MALDI of contaminated samples. We systematically examined the ability to remove measured amounts of contaminants from peptide and protein samples by doping previously pure samples with glycerol and salts. Samples doped with 5 % glycerol and 500 mM sodium were prepared for MALDI-MS analysis using the method described above. 

Although analysis of peptide masses in the total supernatant allows a tentative identification, mass overlap at this resolution may lead to erroneous assignments. For example, signal suppression can lead to low intensity or abolition of certain peptide signals. Since the peptides are tentatively identified on the basis of mass alone, it is prudent to perform PSD analysis to obtain sequence information and confirm the identity. PSD analysis could be performed on some peptides in the total mixture however, it was difficult to obtain sequence information on low... 

Peptides 1000-4000 - Ion suppression - High dynamic range - Sensitivity - MS/MS - High mass resolution... 

ToF Proteins, peptides, lipids, metabolites No upper mass limit high sensitivity Low mass resolution... 

FT-ICR Metabolites, lipids, peptides Highest mass resolution accurate mass Expensive acquisition and maintenance... 

FT-Orbitrap Metabolites, lipids, peptides Very high mass resolution accurate mass leading MS/MS capabilities Imaging sources provided by third-party companies... 

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