Abstract:Absolute protein quantification methods based on molecular mass spectrometry usually require stable isotope-labeled analogous standards for each target protein or peptide under study, which in turn must be certified using natural standards. In this work, we report a direct and accurate methodology based on capLC-ICP-QQQ and online isotope dilution analysis for the absolute and sensitive quantification of intact proteins. The combination of the postcolumn addition of S and a generic S-containing internal standa… Show more
“…This is especially critical in the case of intact protein analysis. Even so, the continuous advances in HPLC together with triple quadrupole ICP‐MS technology head towards the possibility of absolute quantification of both peptides and intact proteins . In fact, if correction of the ICP‐MS sensitivity along the LC gradient is achieved, absolute quantification of several intact proteins in relatively simple mixtures using a single S‐containing generic standard, spiked to the sample prior to the LC‐ICP‐MS analysis, becomes possible.…”
“…Unsuitability of AAA and limitations of CNL to intact protein quantification demand for new strategies for full‐length protein standards characterization and certification. In this vein, proven capabilities of ICP‐MS for the quantitative analysis of intact proteins place this methodology as a feasible alternative, because lack of standards for protein certification result in the need for compound‐independent approaches. In contrast to peptides, large number of amino acids in the protein sequence entails the presence of at least one cysteine or methionine residue.…”
“…In that sense, Fig. shows a model workflow that described the synergic combination of elemental MS, which provides quantitative information, and molecular MS, enabling identity and sequence characterization of the species, as has already been tested for the absolute quantitative profiling of an assayed proteome …”
Mass spectrometry-based approaches have enabled important breakthroughs in quantitative proteomics in the last decades. This development is reflected in the better quantitative assessment of protein levels as well as to understand post-translational modifications and protein complexes and networks. Nowadays, the focus of quantitative proteomics shifted from the relative determination of proteins (ie, differential expression between two or more cellular states) to absolute quantity determination, required for a more-thorough characterization of biological models and comprehension of the proteome dynamism, as well as for the search and validation of novel protein biomarkers. However, the physico-chemical environment of the analyte species affects strongly the ionization efficiency in most mass spectrometry (MS) types, which thereby require the use of specially designed standardization approaches to provide absolute quantifications. Most common of such approaches nowadays include (i) the use of stable isotope-labeled peptide standards, isotopologues to the target proteotypic peptides expected after tryptic digestion of the target protein; (ii) use of stable isotope-labeled protein standards to compensate for sample preparation, sample loss, and proteolysis steps; (iii) isobaric reagents, which after fragmentation in the MS/MS analysis provide a final detectable mass shift, can be used to tag both analyte and standard samples; (iv) label-free approaches in which the absolute quantitative data are not obtained through the use of any kind of labeling, but from computational normalization of the raw data and adequate standards; (v) elemental mass spectrometry-based workflows able to provide directly absolute quantification of peptides/proteins that contain an ICP-detectable element. A critical insight from the Analytical Chemistry perspective of the different standardization approaches and their combinations used so far for absolute quantitative MS-based (molecular and elemental) proteomics is provided in this review.
“…This is especially critical in the case of intact protein analysis. Even so, the continuous advances in HPLC together with triple quadrupole ICP‐MS technology head towards the possibility of absolute quantification of both peptides and intact proteins . In fact, if correction of the ICP‐MS sensitivity along the LC gradient is achieved, absolute quantification of several intact proteins in relatively simple mixtures using a single S‐containing generic standard, spiked to the sample prior to the LC‐ICP‐MS analysis, becomes possible.…”
“…Unsuitability of AAA and limitations of CNL to intact protein quantification demand for new strategies for full‐length protein standards characterization and certification. In this vein, proven capabilities of ICP‐MS for the quantitative analysis of intact proteins place this methodology as a feasible alternative, because lack of standards for protein certification result in the need for compound‐independent approaches. In contrast to peptides, large number of amino acids in the protein sequence entails the presence of at least one cysteine or methionine residue.…”
“…In that sense, Fig. shows a model workflow that described the synergic combination of elemental MS, which provides quantitative information, and molecular MS, enabling identity and sequence characterization of the species, as has already been tested for the absolute quantitative profiling of an assayed proteome …”
Mass spectrometry-based approaches have enabled important breakthroughs in quantitative proteomics in the last decades. This development is reflected in the better quantitative assessment of protein levels as well as to understand post-translational modifications and protein complexes and networks. Nowadays, the focus of quantitative proteomics shifted from the relative determination of proteins (ie, differential expression between two or more cellular states) to absolute quantity determination, required for a more-thorough characterization of biological models and comprehension of the proteome dynamism, as well as for the search and validation of novel protein biomarkers. However, the physico-chemical environment of the analyte species affects strongly the ionization efficiency in most mass spectrometry (MS) types, which thereby require the use of specially designed standardization approaches to provide absolute quantifications. Most common of such approaches nowadays include (i) the use of stable isotope-labeled peptide standards, isotopologues to the target proteotypic peptides expected after tryptic digestion of the target protein; (ii) use of stable isotope-labeled protein standards to compensate for sample preparation, sample loss, and proteolysis steps; (iii) isobaric reagents, which after fragmentation in the MS/MS analysis provide a final detectable mass shift, can be used to tag both analyte and standard samples; (iv) label-free approaches in which the absolute quantitative data are not obtained through the use of any kind of labeling, but from computational normalization of the raw data and adequate standards; (v) elemental mass spectrometry-based workflows able to provide directly absolute quantification of peptides/proteins that contain an ICP-detectable element. A critical insight from the Analytical Chemistry perspective of the different standardization approaches and their combinations used so far for absolute quantitative MS-based (molecular and elemental) proteomics is provided in this review.
“…Very recently, Calderón-Celis et al [48] have reported the application of RP-μHPLC-ICP-QQQ and on-line 34 S isotope dilution analysis for the absolute quantitative analysis of the major toxins comprising the venom proteome of the Mozambique spitting cobra, Naja mossambica . Identification of the toxins eluting along the chromatographic separation was carried out by ESI-MS mass profiling in parallel to the ICP-MS measurements, matching the recorded isotope-averaged molecular masses to the calculated masses for mature Naja spp.…”
Section: Icp-msmentioning
confidence: 99%
“…3.Fig. 3
a Scheme of the parallel hybrid RP-μHPLC-ICP-QQQ with on-line 34 S isotope dilution and LC-ESI-QToF analyses for the absolute quantitative analysis of the major toxins identified by mass profiling in the venom of the Mozambique spitting cobra, Naja mossambica [48]. b Overlay of ESI-QToF protein ( blue trace , left
y-axis ) and ICP-QQQ 32 S ( red trace , right
y-axis ) chromatograms allowed peak correlation of ICP-QQQ and ESI-QToF spectra.…”
In this paper we discuss recent significant developments in the field of venom research, specifically the emergence of top-down proteomic applications that allow achieving compositional resolution at the level of the protein species present in the venom, and the absolute quantification of the venom proteins (the term “protein species” is used here to refer to all the different molecular forms in which a protein can be found. Please consult the special issue of Jornal of Proteomics “Towards deciphering proteomes via the proteoform, protein speciation, moonlighting and protein code concepts” published in 2016, vol. 134, pages 1-202). Challenges remain to be solved in order to achieve a compact and automated platform with which to routinely carry out comprehensive quantitative analysis of all toxins present in a venom. This short essay reflects the authors’ view of the immediate future in this direction for the proteomic analysis of venoms, particularly of snakes.
The advent of soft ionization mass spectrometry‐based proteomics in the 1990s led to the development of a new dimension in biology that conceptually allows for the integral analysis of whole proteomes. This transition from a reductionist to a global‐integrative approach is conditioned to the capability of proteomic platforms to generate and analyze complete qualitative and quantitative proteomics data. Paradoxically, the underlying analytical technique, molecular mass spectrometry, is inherently nonquantitative. The turn of the century witnessed the development of analytical strategies to endow proteomics with the ability to quantify proteomes of model organisms in the sense of “an organism for which comprehensive molecular (genomic and/or transcriptomic) resources are available.” This essay presents an overview of the strategies and the lights and shadows of the most popular quantification methods highlighting the common misuse of label‐free approaches developed for model species' when applied to quantify the individual components of proteomes of nonmodel species (In this essay we use the term “non‐model” organisms for species lacking comprehensive molecular (genomic and/or transcriptomic) resources, a circumstance that, as we detail in this review‐essay, conditions the quantification of their proteomes.). We also point out the opportunity of combining elemental and molecular mass spectrometry systems into a hybrid instrumental configuration for the parallel identification and absolute quantification of venom proteomes. The successful application of this novel mass spectrometry configuration in snake venomics represents a proof‐of‐concept for a broader and more routine application of hybrid elemental/molecular mass spectrometry setups in other areas of the proteomics field, such as phosphoproteomics, metallomics, and in general in any biological process where a heteroatom (i.e., any atom other than C, H, O, N) forms integral part of its mechanism.
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