Peptide ions

These authors also mention some shortcomings that should be borne in mind, in particular, that some peptides observed were from the autolysis of trypsin, the digestion agent, and from contaminants such as human keratin, while some peptide ions did not produce interpretable MS-MS spectra. 

Geromanos, S., Freckleton, G., and Tempst, P., Tuning of an electrospray ionization source for maximum peptide-ion transmission into a mass spectrometer, Anal. Chem., 72, 777, 2000. 

Figure 2.3. A. Mass spectrometer consisting of an ionization source, a mass analyzer and an ion detector. The mass analyzer shown is a time-of -flight (TOF) mass spectrometer. Mass-to-charge (m/z) ratios are determined hy measuring the amount of time it takes an ion to reach the detector. B. Tandem mass spectrometer consisting of an ion source, a first mass analyzer, a collision cell, a second mass analyzer and a detector. The first mass analyzer is used to choose a particular peptide ion to send to the collision cell where the peptide is fragmented. The mass of the spectrum of fragments is determined in the second mass analyzer and is diagnostic of the amino acid sequence of the peptide. Figure adapted from Yates III (2000).

Brown, R. S. Lennon, J. J. Sequence-specific fragmentation of matrix-assisted laser-desorbed protein/peptide ions. Anal. Chem. 1995,67,3990-3999. 

With TOF, the ions from an ion source are accelerated linearly down a chamber containing an electrical field. The flight chamber is at very low pressure that facilitates the flight of the peptide ions with minimal collisions with other molecules. The ions travel in a linear trajectory until they impact a detector at the other end of the tube. The heavier ions travel more slowly than the lower molecular weight ions and reach the detector later. Hence, TOF analyzers derive their name from the concept that the time of flight of an ion is related to its m/z ratio and velocity within a fixed distance. Linear mode TOF analyzers contain single chambers and are not favored for proteomics applications because of their lower mass accuracy. 

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. 

Figure 3.3. Nomenclature of peptide ions chemical structure of b, c, z, and y product io...

Figure 6.5. Nomenclature of peptide ions resulting from backbone fragmentation.

Fig. 5. Fragmentation nomenclature of peptides. Bond breakages of all bonds of the peptide backbone have a systematic name (I). When fragmenting multiply charged peptide ions the peptide bond breaks preferentially since it is among the most labile bonds and only relatively low collision energies are involved (II).

Continuous ion series are often generated when multiply charged peptide ions are fragmented. The problem in de novo sequencing with electrospray tandem mass spectrometry lies in minimizing the error rate of the interpretation. There are two different approaches to this problem ... 

It would be complicating if not impossible having to obtain sequence information from such a spectrum without rules to follow. [138-141,146,147] The most abundant ions obtained from the fragmenting peptide ion usually belong to six series named a, b, and c if the proton (charge) is kept in the A-terminus or x, y, and z, respectively, where the proton is located in the C-terminal part. Within each se-... 

Pan, Y. Cotter, R.J. Measurement of Initial Translational Energies of Peptide Ions in Laser Desorption/Ionization-MS. Org. Mass Spectrom. 1992, 27, 3-8. 

Example The reduced sample consumption of nanoESI allows for the sequencing of the peptides (Chap. 9.4.7) obtained by tryptic digestion of only 800 fmol of the protein bovine semm albumin (BSA, Fig. 11.6). [66] The experiment depicted below requires each of the BSA-derived peptide ions in the full scan spectrum to be subjected to fragment ion analysis by means of CID-MS/MS on a triple quadrupole instmment (Chaps. 2.12 and 4.4.5). 

Fig. 11.6. Peptide sequencing by nanoESI-CID-MS/MS from a tryptic digest of bovine serum albumin (BSA) 800 fmol of BSA were used, (a) Eull scan spectrum, (b) fragmentation of the selected doubly charged peptide ion at m/z 740.5. Adapted from Ref. [66] by permission. Nature Publishing Group, 1996.

More recent work revealed the importance of gas phase proton transfer reactions. [91-94] This implies that multiply charged peptide ions do not exist as preformed ions in solution, but are generated by gas phase ion-ion reactions (Chap. 11.4.4). The proton exchange is driven by the difference in proton affinities (PA, Chap. 2.11) of the species encountered, e.g., a protonated solvent molecule of low PA will protonate a peptide ion with some basic sites left. Under equilibrium conditions, the process would continue until the peptide ion is saturated with protons, a state that also marks its maximum number of charges. 

Many of the advantages that MALDI offers for peptide analysis are equally applicable to proteins. Protein analysis is similar to peptide analysis, in which ionization usually occurs through the addition of one, two, or three protons. However, since proteins are significantly bigger than peptides, ion detection is typically less efficient. Therefore, while peptides are measured at the femtomole or even attomole level with MALDI, proteins are usually measured at the high femtomole to low picomole level. 

Scheme 1 The Expected Fragmentation Pattern of Protonated Peptide Ions and the Nomenclature of the Amino Acid Sequence Fragment Ions for Determining Amino Acid Sequences...

The sample to be analyzed is introduced to the ESI source by means of a flow stream from an HPLC instrument. The sample flows through a stainless-steel needle and then, sprays out in the form of a mist whose droplets hold peptide ions and mobile phase of HPLC. Peptide ions are separated from the mobile phase and subsequently, transferred into a mass analyzer either by a heated capillary or a curtain of nitrogen gas. Desolvation process can be carried out by a vacuum system. 

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