Characterization of high molecular weight multimeric states of human haptoglobin and hemoglobin‐based oxygen carriers by high‐mass MALDI MS

Pimenova, Tatiana; Pereira, Cláudia; Schaer, Dominik J.; Zenobi, Renato

doi:10.1002/jssc.200800625

Cited by 46 publications

(55 citation statements)

References 41 publications

(61 reference statements)

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“…The application of blood fluids to size exclusion chromatography does capture low molecular mass peptides (Longo et al, 2009) but failed to yield the best signal to noise ratio or number of peptides in direct comparisons with other methods (Tucholska et al, 2007;Williams et al, 2007). Size exclusion was used to reveal plasma protein multimeric and other complexes by MALDI (Pimenova et al, 2009). Size exclusion has been coupled to strong cation exchange resin for a two-dimensional analysis of serum peptides and resulted in thousands of identified peptides (Hu et al, 2009a).…”

Section: Ultrafiltration and Size Exclusionmentioning

confidence: 99%

Mass spectrometry of peptides and proteins from human blood

Zhu

Bowden

Zhang

et al. 2010

Mass Spectrometry Reviews

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It is difficult to convey the accelerating rate and growing importance of mass spectrometry applications to human blood proteins and peptides. Mass spectrometry can rapidly detect and identify the ionizable peptides from the proteins in a simple mixture and reveal many of their post-translational modifications. However, blood is a complex mixture that may contain many proteins first expressed in cells and tissues. The complete analysis of blood proteins is a daunting task that will rely on a wide range of disciplines from physics, chemistry, biochemistry, genetics, electromagnetic instrumentation, mathematics and computation. Therefore the comprehensive discovery and analysis of blood proteins will rank among the great technical challenges and require the cumulative sum of many of mankind's scientific achievements together. A variety of methods have been used to fractionate, analyze and identify proteins from blood, each yielding a small piece of the whole and throwing the great size of the task into sharp relief. The approaches attempted to date clearly indicate that enumerating the proteins and peptides of blood can be accomplished. There is no doubt that the mass spectrometry of blood will be crucial to the discovery and analysis of proteins, enzyme activities, and post-translational processes that underlay the mechanisms of disease. At present both discovery and quantification of proteins from blood are commonly reaching sensitivities of ∼1 ng/mL.

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Section: Ultrafiltration and Size Exclusionmentioning

confidence: 99%

Mass spectrometry of peptides and proteins from human blood

Zhu

Bowden

Zhang

et al. 2010

Mass Spectrometry Reviews

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“…Novel detector technologies allow measurements of analytes up to several MDa and provide the basis for studying immunocomplexes and protein multimers [10,11]. With increasing protein mass, signals corresponding to multiply charged species emerge in the MALDI mass spectra.…”

Section: Introductionmentioning

confidence: 99%

Compelling Advantages of Negative Ion Mode Detection in High-Mass MALDI-MS for Homomeric Protein Complexes

Mädler

Barylyuk

Erba

et al. 2011

J. Am. Soc. Mass Spectrom.

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Chemical cross-linking in combination with high-mass MALDI mass spectrometry allows for the rapid identification of interactions and determination of the complex stoichiometry of noncovalent protein-protein interactions. As the molecular weight of these complexes increases, the fraction of multiply charged species typically increases. In the case of homomeric complexes, signals from multiply charged multimers overlap with singly charged subunits. Remarkably, spectra recorded in negative ion mode show lower abundances of multiply charged species, lower background, higher reproducibility, and, thus, overall cleaner spectra compared with positive ion mode spectra. In this work, a dedicated high-mass detector was applied for measuring highmass proteins (up to 200 kDa) by negative ion mode MALDI-MS. The influences of sample preparation and instrumental parameters were carefully investigated. Relative signal integrals of multiply charged anions were relatively independent of any of the examined parameters and could thus be approximated easily for the spectra of cross-linked complexes. For example, the fraction of doubly charged anions signals overlapping with the signals of singly charged subunits could be more precisely estimated than in positive ion mode. Sinapinic acid was found to be an excellent matrix for the analysis of proteins and cross-linked protein complexes in both ion modes. Our results suggest that negative ion mode data of chemically cross-linked protein complexes are complementary to positive ion mode data and can in some cases represent the solution phase situation better than positive ion mode.

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“…During MALDI ionization, most of the species present in the sample appear as singly charged pseudo-molecular ions in the spectra, greatly simplifying the data interpretation for complex mixtures. In combination with novel detector technologies, the mass range has been significantly extended, to more than 1 MDa, allowing sensitive detection of, for example, immunocomplexes or protein multimers [7,8]. Among the most common cross-linking agents are homobifunctional N-hydroxy succinimide (NHS) esters, which were introduced more than 30 years ago and shown to be highly aminereactive [9].…”

mentioning

confidence: 99%

Does chemical cross-linking with NHS esters reflect the chemical equilibrium of protein-protein noncovalent interactions in solution?

Mädler

Seitz

Zenobi

2010

J. Am. Soc. Mass Spectrom.

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Chemical cross-linking in combination with mass spectrometry has emerged as a powerful tool to study noncovalent protein complexes. Nevertheless, there are still many questions to answer. Does the amount of detected cross-linked complex correlate with the amount of protein complex in solution? In which concentration and affinity range is specific cross-linking possible? To answer these questions, we performed systematic cross-linking studies with two complexes, using the N-hydroxysuccinimidyl ester disuccinimidyl suberate (DSS): (1) NCoA-1 and mutants of the interacting peptide STAT6Y, covering a K D range of 30 nM to Ͼ25 M, and (2) ␣-thrombin and basic pancreatic trypsin inhibitor (BPTI), a system that shows a bufferdependent K D value between 100 and 320 M. Samples were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). For NCoA-1•STAT6Y, a good correlation between the amount of cross-linked species and the calculated fraction of complex present in solution was observed. Thus, chemical cross-linking in combination with MALDI-MS can be used to rank binding affinities. For the mid-affinity range up to about K D Ϸ 25 M, experiments with a nonbinding peptide and studies of the concentration dependence showed that only specific complexes undergo cross-linking with DSS. To study in which affinity range specific cross-linking can be applied, the weak ␣-thrombin•BPTI complex was investigated. We found that the detected complex is a nonspecifically cross-linked species. Consequently, based on the experimental approach used in this study, chemical cross-linking is not suitable for studying low-affinity complexes with K D ӷ 25 M. (J Am Soc Mass Spectrom 2010Spectrom , 21, 1775Spectrom -1783

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Characterization of high molecular weight multimeric states of human haptoglobin and hemoglobin‐based oxygen carriers by high‐mass MALDI MS

Cited by 46 publications

References 41 publications

Mass spectrometry of peptides and proteins from human blood

Mass spectrometry of peptides and proteins from human blood

Compelling Advantages of Negative Ion Mode Detection in High-Mass MALDI-MS for Homomeric Protein Complexes

Does chemical cross-linking with NHS esters reflect the chemical equilibrium of protein-protein noncovalent interactions in solution?

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