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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

Native LESA mass spectra of hemoglobin: (a) glass substrate, Orbitrap mass analyzer; (b) glass substrate, Q-TOF mass analyzer; (c) PVDF substrate, Orbitrap mass analyzer; (d) PVDF substrate, Q-TOF mass analyzer
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Fig3: Native LESA mass spectra of hemoglobin: (a) glass substrate, Orbitrap mass analyzer; (b) glass substrate, Q-TOF mass analyzer; (c) PVDF substrate, Orbitrap mass analyzer; (d) PVDF substrate, Q-TOF mass analyzer

Mentions: Figure 3 shows the results obtained following LESA MS of hemoglobin standard dried onto glass and PVDF substrates. For comparison, the direct infusion mass spectra of the protein are shown in Figure 2c and d. As described above, in its native state hemoglobin exists as a noncovalent tetramer of the form (αβ)24H. Figure 3a shows the mass spectrum obtained on the Orbitrap following LESA of the glass substrate. The spectrum is characterized by noncovalent αH and βH monomers, noncovalent heterodimers (αβ)2H, and those with a single heme (αβ)H. Heme-deficient dimers have been reported in previous examples of native hemoglobin analysis [33, 34]. It is notable that the βH monomer was of significantly lower abundance than the αH monomer. Also observed are peaks corresponding to beta globin with a mass shift Δm +32 Da. This observation has been reported previously and has been speculated to be due to oxidation of the beta chain [33]. Tetramers were not observed. Similar results were obtained following LESA of the PVDF substrate (Figure 3c), although the relative abundance of the dimers was lower. The LESA MS results are in good agreement with the direct infusion ESI results (Figure 2). The relative abundance of the dimers was higher following direct infusion ESI and no dominant peaks at m/z <800 were observed.Figure 3


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)

Native LESA mass spectra of hemoglobin: (a) glass substrate, Orbitrap mass analyzer; (b) glass substrate, Q-TOF mass analyzer; (c) PVDF substrate, Orbitrap mass analyzer; (d) PVDF substrate, Q-TOF mass analyzer
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Native LESA mass spectra of hemoglobin: (a) glass substrate, Orbitrap mass analyzer; (b) glass substrate, Q-TOF mass analyzer; (c) PVDF substrate, Orbitrap mass analyzer; (d) PVDF substrate, Q-TOF mass analyzer
Mentions: Figure 3 shows the results obtained following LESA MS of hemoglobin standard dried onto glass and PVDF substrates. For comparison, the direct infusion mass spectra of the protein are shown in Figure 2c and d. As described above, in its native state hemoglobin exists as a noncovalent tetramer of the form (αβ)24H. Figure 3a shows the mass spectrum obtained on the Orbitrap following LESA of the glass substrate. The spectrum is characterized by noncovalent αH and βH monomers, noncovalent heterodimers (αβ)2H, and those with a single heme (αβ)H. Heme-deficient dimers have been reported in previous examples of native hemoglobin analysis [33, 34]. It is notable that the βH monomer was of significantly lower abundance than the αH monomer. Also observed are peaks corresponding to beta globin with a mass shift Δm +32 Da. This observation has been reported previously and has been speculated to be due to oxidation of the beta chain [33]. Tetramers were not observed. Similar results were obtained following LESA of the PVDF substrate (Figure 3c), although the relative abundance of the dimers was lower. The LESA MS results are in good agreement with the direct infusion ESI results (Figure 2). The relative abundance of the dimers was higher following direct infusion ESI and no dominant peaks at m/z <800 were observed.Figure 3

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