Proteins intact mass

A growing number of researchers are focusing on the use of top-down proteomics, a relatively new approach compared to bottom-up, in which structure of proteins is studied through measurement of their intact mass followed by direct ion dissociation in the gas phase. The main advantages over the bottom-up approach are that higher sequence coverage is obtained, it permits... 

Figure 2. Assessment of the complexity of a protein mixture by combined strategies. A protein mixture from mouse urine was analysed by ion exchange chromatography (charge based separation) and by mass spectrometry of the intact proteins. Both methods allude to the complexity of the mixture. However, when the proteins separated by chromatography were analysed by mass spectrometry, the largest peak was demonstrated to contain two proteins. Moreover, the chromatographic separation indicated that there were two protein peaks that contained proteins of mass 18693Da. The techniques give orthogonal and complementary views of the complexity of the analyte.

Figure 4 Using mass spectrometry to confirm identity and disuifide bond formation for a CXCL12 mutant. (A) The intact mass spectrum of foided and oxidized CXCL12 S-1 S4V with the charge state enveiope inset. (B) The denatured and reduced protein, compared to the oxidized, and refoided protein the mass increased in size by 4 Da with aii four cysteines being reduced and the charge state enveiope has shifted to a iower m/z.

Biology TOF-MS (ESI) Intact protein molecular mass determination... 

Depending on the size and properties of the protein, 2 to SOpmol of purified protein is needed for a complete and comprehensive molecular analysis by multiple MALDI-MS experiments, including intact mass determination, several peptide mass mapping experiments, and MS/MS analysis of individual peptides (see below). 

Intact mass determination is a very useful first step towards the full characterization of proteins (Figure 3.4), and the purified protein should therefore be analyzed by MALDI-MS for determination of the molecular mass. Gel electrophoresis will often be used as the final stage of protein purification, depending on the purification procedure used. If not, it is advantageous to monitor the protein purification... 

In order to evaluate the proteomic profile of whole cells and to correctly identify proteins based solely on the intact mass, advanced instrumentation offering high mass accuracy is necessary. The application of Fourier transform mass spectrometry (FTMS) to intact cell analysis allowed for unambiguous identification of biomarker proteins due to the high resolution and mass... 

A second advantage of the LC-ESI-MS approach is the increased mass accn-racy of the measurement, making possible the detection of small mass differences between proteins. These mass differences can be eqirivalent to single-nucleotide polymorphisms (SNP) mutations or post-translational modifications. Recent work by McFarland and coworkers (2014) best illustrates the implementation of LC-(ESI)-MS (intact protein mass) and MS/MS top-down analyses to bacteria differentiation at the strain level. Proteins extracted from bacterial samples of Salmonella typhimurium (strain LT2) and S. heidelberg (strain A3 9) were first separated by reversed-phase (RP) LC and the eluent analyzed directly by ESI-MS using Q-TOF MS system (operated in the full-scan mode or MS). Following the data processing... 

FIGURE 8.16 Three views of an unknown protein obtained by MALDI-TOF mass spectrometry (a) linearmode mass spectrum of the intact protein, (b) mass map of the tryptic peptides derived from the unknown protein, and (c) PSD mass spectrum of peptide I from the mixture in (b). (Reprinted with permission from reference I i). 

Technologies that offer the opportunity to use MS for intact protein mass determination allow rapid assessment of signal peptide cleavage sites and post-translational heterogeneity. A well-resolved mass spectrum of an intact protein defines its native covalent profile as well as its associated heterogeneity. A comprehensive mass spectrometric approach that integrates both intact protein molecular mass measurement (top-down) and proteolytic fragment identification (bottom-up) would represent a powerful approach for proteome/ protein characterization. 

In another application, UHPLC-MS technology was developed for rapid comparison of a candidate biosimilar to an innovator monoclonal antibody (mAb) (37). In this study, UHPLC-MS was developed for rapid verification of identity and characterization of sequence variants and posttranslational modifications (PTMs) for mAb products. Although the biosimilar product is expected to have the same amino acid sequence and modifications as the innovator s product, the observed intact mass by UHPLC-MS was different for the biosimilar compared to the innovator protein. Peptide mapping using UHPLC-MS/MS (38) revealed that the mass difference between the biosimilar and the innovator s product was due to a two amino acid residue variance in the heavy chain sequence of the biosimilar (Figure 8.6). 

The ions so produced are separated by their mass-to-charge (m/z) ratios. For peptides and proteins, the intact molecules become protonated with a number (n) of protons (H+). Thus, instead of the true molecular mass (M), molecular ions have a mass of [M + uH]. More importantly, the ion has n positive charges resulting from addition of the n protons [M + uH]". Since the mass spectrometer does not measure mass directly but, rather, mass-to-charge (m/z) ratio, the measured m/z value is [M + uH]/u. This last value is less than the true molecular mass, depending on the value of n. If the ion of true mass 20,000 Da carries 10 protons, for example, then the m/z value measured would be (20,000 + 10)/10 = 2001. 

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