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Native Liquid Extraction Surface Analysis Mass Spectrometry: Analysis of Noncovalent Protein Complexes Directly from Dried Substrates.

Martin NJ, Griffiths RL, Edwards RL, Cooper HJ - J. Am. Soc. Mass Spectrom. (2015)

Bottom Line: Holomyoglobin, in which apomyoglobin is noncovalently bound to the prosthetic heme group, was observed following LESA mass spectrometry of myoglobin dried onto glass and polyvinylidene fluoride surfaces.Heme-bound dimers and monomers were also observed.The 'contact' LESA approach was particularly suitable for the analysis of hemoglobin tetramers from DBS.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.

ABSTRACT
Liquid extraction surface analysis (LESA) mass spectrometry is a promising tool for the analysis of intact proteins from biological substrates. Here, we demonstrate native LESA mass spectrometry of noncovalent protein complexes of myoglobin and hemoglobin from a range of surfaces. Holomyoglobin, in which apomyoglobin is noncovalently bound to the prosthetic heme group, was observed following LESA mass spectrometry of myoglobin dried onto glass and polyvinylidene fluoride surfaces. Tetrameric hemoglobin [(αβ)2(4H)] was observed following LESA mass spectrometry of hemoglobin dried onto glass and polyvinylidene fluoride (PVDF) surfaces, and from dried blood spots (DBS) on filter paper. Heme-bound dimers and monomers were also observed. The 'contact' LESA approach was particularly suitable for the analysis of hemoglobin tetramers from DBS.

No MeSH data available.


Related in: MedlinePlus

Direct infusion native ESI mass spectra of (a) myoglobin obtained with Orbitrap mass analyzer; (b) myoglobin obtained with Q-TOF mass analyzer; (c) hemoglobin obtained with Orbitrap mass analyzer; (d) hemoglobin obtained with Q-TOF mass analyzer
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Fig2: Direct infusion native ESI mass spectra of (a) myoglobin obtained with Orbitrap mass analyzer; (b) myoglobin obtained with Q-TOF mass analyzer; (c) hemoglobin obtained with Orbitrap mass analyzer; (d) hemoglobin obtained with Q-TOF mass analyzer

Mentions: Figure 1 shows the results obtained following LESA mass spectrometry of myoglobin dried onto glass and PVDF surfaces. Figure 1a and c show results obtained on the Orbitrap and Figure 1b and d on the Q-TOF. LESA MS of myoglobin on glass resulted in detection of the noncovalent myoglobin–heme complex (holomyoglobin) MbH. Dominant peaks in the Orbitrap mass spectrum correspond to the 9+, 8+, and 7+ charge states of holomyoglobin at m/z 1952.78, 2196.75, and 2510.42. (Stated m/z values from Orbitrap data are for the most abundant isotopic peak). The Q-TOF spectrum showed peaks corresponding to the 9+, 8+, 7+, and 6+ charge states of MbH at m/z 1953, 2197, 2510, and 2927. (Stated m/z values from Q-TOF data correspond to the maximum peak height). Differences in the observed charge states are likely due to the capillary temperatures of the Orbitrap and the Q-TOF: higher temperatures can favor the formation of higher charge states [29]. Higher temperatures can lead to greater unfolding of the protein, thus exposing more basic amino acid residues (with subsequent protonation). Additionally, higher temperatures may lead to more effective desolvation during ionization [10]. Heating during desolvation helps eliminate neutral molecules that become deposited on the protein, which may prevent it from obtaining high charge states [9, 29]. Peaks corresponding to apo-myoglobin were observed at low abundance in both the Q-TOF mass spectrum and the Orbitrap spectrum. Notably, the peaks corresponding to apomyoglobin are of higher abundance in the Q-TOF spectrum compared with those in the Orbitrap mass spectrum. Given that the samples were prepared identically and that higher temperatures were encountered in the Orbitrap (see above), it is likely that this observation is the result of differences in ion transfer parameters in the two instruments. There are numerous singly and doubly charged peaks below m/z 1200 in the Orbitrap mass spectrum, which correspond to a polymeric background ion with a repeating unit of mass 44 Da. For comparison, the direct infusion mass spectra of myoglobin obtained on the Orbitrap and Q-TOF instruments are shown in Figure 2a and b. The peaks corresponding to noncovalent complexes and the charge state distributions match those observed in the LESA mass spectra. There are no peaks corresponding to apomyoglobin, suggesting that dissociation occurs in the drying and/or extraction process. There are also no dominant peaks at m/z <1200, suggesting that those peaks observed in the LESA mass spectrum may originate from the glass substrate.Figure 1


Native Liquid Extraction Surface Analysis Mass Spectrometry: Analysis of Noncovalent Protein Complexes Directly from Dried Substrates.

Martin NJ, Griffiths RL, Edwards RL, Cooper HJ - J. Am. Soc. Mass Spectrom. (2015)

Direct infusion native ESI mass spectra of (a) myoglobin obtained with Orbitrap mass analyzer; (b) myoglobin obtained with Q-TOF mass analyzer; (c) hemoglobin obtained with Orbitrap mass analyzer; (d) hemoglobin obtained with Q-TOF mass analyzer
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4494149&req=5

Fig2: Direct infusion native ESI mass spectra of (a) myoglobin obtained with Orbitrap mass analyzer; (b) myoglobin obtained with Q-TOF mass analyzer; (c) hemoglobin obtained with Orbitrap mass analyzer; (d) hemoglobin obtained with Q-TOF mass analyzer
Mentions: Figure 1 shows the results obtained following LESA mass spectrometry of myoglobin dried onto glass and PVDF surfaces. Figure 1a and c show results obtained on the Orbitrap and Figure 1b and d on the Q-TOF. LESA MS of myoglobin on glass resulted in detection of the noncovalent myoglobin–heme complex (holomyoglobin) MbH. Dominant peaks in the Orbitrap mass spectrum correspond to the 9+, 8+, and 7+ charge states of holomyoglobin at m/z 1952.78, 2196.75, and 2510.42. (Stated m/z values from Orbitrap data are for the most abundant isotopic peak). The Q-TOF spectrum showed peaks corresponding to the 9+, 8+, 7+, and 6+ charge states of MbH at m/z 1953, 2197, 2510, and 2927. (Stated m/z values from Q-TOF data correspond to the maximum peak height). Differences in the observed charge states are likely due to the capillary temperatures of the Orbitrap and the Q-TOF: higher temperatures can favor the formation of higher charge states [29]. Higher temperatures can lead to greater unfolding of the protein, thus exposing more basic amino acid residues (with subsequent protonation). Additionally, higher temperatures may lead to more effective desolvation during ionization [10]. Heating during desolvation helps eliminate neutral molecules that become deposited on the protein, which may prevent it from obtaining high charge states [9, 29]. Peaks corresponding to apo-myoglobin were observed at low abundance in both the Q-TOF mass spectrum and the Orbitrap spectrum. Notably, the peaks corresponding to apomyoglobin are of higher abundance in the Q-TOF spectrum compared with those in the Orbitrap mass spectrum. Given that the samples were prepared identically and that higher temperatures were encountered in the Orbitrap (see above), it is likely that this observation is the result of differences in ion transfer parameters in the two instruments. There are numerous singly and doubly charged peaks below m/z 1200 in the Orbitrap mass spectrum, which correspond to a polymeric background ion with a repeating unit of mass 44 Da. For comparison, the direct infusion mass spectra of myoglobin obtained on the Orbitrap and Q-TOF instruments are shown in Figure 2a and b. The peaks corresponding to noncovalent complexes and the charge state distributions match those observed in the LESA mass spectra. There are no peaks corresponding to apomyoglobin, suggesting that dissociation occurs in the drying and/or extraction process. There are also no dominant peaks at m/z <1200, suggesting that those peaks observed in the LESA mass spectrum may originate from the glass substrate.Figure 1

Bottom Line: Holomyoglobin, in which apomyoglobin is noncovalently bound to the prosthetic heme group, was observed following LESA mass spectrometry of myoglobin dried onto glass and polyvinylidene fluoride surfaces.Heme-bound dimers and monomers were also observed.The 'contact' LESA approach was particularly suitable for the analysis of hemoglobin tetramers from DBS.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.

ABSTRACT
Liquid extraction surface analysis (LESA) mass spectrometry is a promising tool for the analysis of intact proteins from biological substrates. Here, we demonstrate native LESA mass spectrometry of noncovalent protein complexes of myoglobin and hemoglobin from a range of surfaces. Holomyoglobin, in which apomyoglobin is noncovalently bound to the prosthetic heme group, was observed following LESA mass spectrometry of myoglobin dried onto glass and polyvinylidene fluoride surfaces. Tetrameric hemoglobin [(αβ)2(4H)] was observed following LESA mass spectrometry of hemoglobin dried onto glass and polyvinylidene fluoride (PVDF) surfaces, and from dried blood spots (DBS) on filter paper. Heme-bound dimers and monomers were also observed. The 'contact' LESA approach was particularly suitable for the analysis of hemoglobin tetramers from DBS.

No MeSH data available.


Related in: MedlinePlus