Interference-Free Proteome Quantification with MS/MS-based Isobaric Isotopologue Detection

Bamberger, Casimir; Pankow, Sandra; Park, Sung Kyu Robin; Yates, John R.

doi:10.1021/pr401035z

Cited by 24 publications

(33 citation statements)

References 22 publications

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“…Although dim-4- to dim-6-YGGFLR share a common fragmentation pathway releasing the derivatizing groups as N,N-dimethyl lactams (Scheme 7), the increased distance between the dimethylamino group and the amide bond formed upon the peptide derivatization leads to a discrete increase in the stability of intact precursors from dim-5- to dim-6-YGGFLR, shown as the positive deviation (Figure 7). This agrees with the observation that the ε-dimethylamino group on the lysine side chain is a passive mass tag (Figure S2e), stable to collisional fragmentation in the gas phase [10, 11]. …”

Section: Resultssupporting

confidence: 90%

“…In line with recent advancements in the utilization of ultra-high resolving MS for proteomics, differences in mass defects of carbon, hydrogen, oxygen, and nitrogen isotopes have been exploited [10, 11, 16-19]. The scope of isobaric mass tagging is thus being expanded, opening exciting new opportunities in proteome quantitation.…”

Section: Introductionmentioning

confidence: 99%

“…Both works use Lys-C for protein digestion so that each resulting peptide carries two derivatizing groups. It is interesting to note that there are more derivatized y ions than b ions available to be used for quantitation [10, 11]; in other words the ε-dimethylamino group on the lysine side chain at the peptide C-terminus is more stable than the α-dimethylamino group at the N-terminus.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Peptide Dimethylation: Fragmentation Control via Distancing the Dimethylamino Group

McShane

Shen

Castillo

et al. 2014

J. Am. Soc. Mass Spectrom.

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Direct reductive methylation of peptides is a common method for quantitative proteomics. It is an active derivatization technique; with participation of the dimethylamino group, the derivatized peptides preferentially release intense a1 ions. The advantageous generation of a1 ions for quantitative proteomic profiling, however, is not desirable for targeted proteomic quantitation using multiple reaction monitoring mass spectrometry; this mass spectrometric method prefers the derivatizing group to stay with the intact peptide ions and multiple fragments as passive mass tags. This work investigated collisional fragmentation of peptides whose amine groups were derivatized with five linear ω-dimethylamino acids, from 2-(dimethylamino)-acetic acid to 6-(dimethylamino)-hexanoic acid. Tandem mass spectra of the derivatized tryptic peptides revealed different preferential breakdown pathways. Together with energy resolved mass spectrometry, it was found that shutting down the active participation of the terminal dimethylamino group in fragmentation of derivatized peptides is possible. However, it took a separation of five methylene groups between the terminal dimethylamino group and the amide formed upon peptide derivatization. For the first time, the gas-phase fragmentation of peptides derivatized with linear ω-dimethylamino acids of systematically increasing alkyl chain lengths is reported.

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Section: Resultssupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Peptide Dimethylation: Fragmentation Control via Distancing the Dimethylamino Group

McShane

Shen

Castillo

et al. 2014

J. Am. Soc. Mass Spectrom.

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“…Mammalian tissues and biofluids, however, are not as easily metabolically labeled and seemingly are not amenable to the strategy (44 -46). For such samples we envision the development of mass defectbearing chemical tags (47)(48)(49)(50). Moreover, these NeuCode labels, unlike the amino acid reagents used herein, could be designed without the use of deuterium.…”

Section: Discussionmentioning

confidence: 99%

NeuCode Labels for Relative Protein Quantification

Merrill

Hebert

MacGilvray

et al. 2014

Molecular & Cellular Proteomics

103

101

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We describe a synthesis strategy for the preparation of lysine isotopologues that differ in mass by as little as 6 mDa. We demonstrate that incorporation of these molecules into the proteomes of actively growing cells does not affect cellular proliferation, and we discuss how to use the embedded mass signatures (neutron encoding (NeuCode)) for multiplexed proteome quantification by means of high-resolution mass spectrometry. NeuCode SILAC amalgamates the quantitative accuracy of SILAC with the multiplexing of isobaric tags and, in doing so, offers up new opportunities for biological investigation. Large-scale technologies for the comparative analysis of proteomes have become essential for modern biology and medicine (1, 2). To satisfy this increasing demand and boost statistical power, parallel processing of proteomes (i.e. multiplexing) is key. The ground was broken in this field about two decades ago by advances in both MS and stable isotope labeling (3-5). Since then, two distinct strategies have emerged, each with its own strengths and weaknesses. The first approach, stable isotope labeling by amino acids in cell culture (SILAC), 1 metabolically incorporates labeled amino acids into proteins and is considered the gold standard (6 -8). SILAC quantification provides unmatched accuracy, but simultaneous comparison of more than three proteomes, although possible, is not practical for most global studies (9, 10). A second, and increasingly popular, method is to chemically modify peptides originating from up to 10 different sources (a 3-to 5-fold boost in throughput over SILAC) with isobaric reagents (e.g. TMT or iTRAQ) (11)(12)(13)(14). The escalated throughput afforded by this strategy is, for many applications, essential; however, multiplexing via isobaric tagging comes at the cost of quantitative accuracy (15-17). Furthermore, because each sample is handled independently prior to labeling, systematic and random variation that occurs during sample processing cannot be accounted for as it is with metabolic labeling. Thus, experimenters designing a quantitative proteomics experiment must choose between accuracy and throughput.Recently we described a new approach that blends the SILAC and isobaric tagging methods (18). The strategy, neutron encoding (NeuCode), relies on the mass defects of atoms and their isotopes (19). In studies using two isotopologues of lysine, differing by 36 mDa, NeuCode SILAC quantified proteins as well as traditional SILAC, but it allowed deeper proteome coverage. NeuCode harnesses the exceptional resolving power of modern FT-MS systems so that quantitative information is only revealed by high-resolution scanning when desired, in either MS or tandem MS scans (20,21).Owing to the lack of suitable lysine isotopologues, our initial work with NeuCode SILAC offered only duplex quantification; consequently, we could only predict the utility of NeuCode SILAC (18). To test our supposition that NeuCode SILAC has the potential to combine the benefits of traditional SILAC and

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“…For samples such as primary cells or tissues, the more practical approach is isobaric tandem mass tagging of peptides using, for instance, TMT (tandem mass tags) or iTRAQ (isobaric tags for relative and absolute quantification) reagents. [41][42][43][44] This method uses isotope-labeled amine-reactive chemical tags to label peptides derived from distinct samples ( Figure 5A). Most laboratories analyze up to 6 samples; however, recent advancements SILAC Cells can be cultured in conditions under which they are dependent on exogenous supplies of amino acids that are either unlabeled ( 12 C and 14 N, light) or stable isotope-labeled ( 13 C, 15 N and sometimes 2 H/deuterium) ( Figure 4C).…”

Section: Tandem Mass Taggingmentioning

confidence: 99%

Current Trends and Future Perspectives of State-of-the-Art Proteomics Technologies Applied to Cardiovascular Disease Research

Singh

Aikawa

2016

Circ J

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The ongoing development of MS is in part influenced by the drive to identify and quantify as many proteins as possible in a given sample. 1,15-17 A major challenge in proteomics, however, is that the dynamic range of a typical deep sequencing analysis (5-6 orders in magnitude) does not reflect the more extensive dynamic range of protein abundance that is as extensive as 12 orders in plasma. 18-20 In addition, cytoskeletal and extracellular matrix proteins dominate samples, masking the less abundant transcription factors and signaling molecules that are usually of interest. 1,18,19 Depletion of these highly abundant proteins, such as albumin from plasma, is required to sequence deep into the proteome ( Figure 1C). 21-23 Also, the less abundant proteins can be enriched by cellular fractionation methods such as those that separate cellular compartments or those that use immunopurification strategies ( Figure 1C). 24, 25 The dynamic range capabilities of MS continually improve, but the identification and quantification of low-abundance proteins still depend on sample preparation procedures that shift these low signals into the detection range of the instrument either by depletion and/or enrichment approaches.any developments in the field of proteomics have come from the yeast model system 1-3 and from cancer research ( Figure 1A). In addition, immortalized cells (eg, HeLa, HEK293T, RAW264.7, THP-1) have provided mass spectrometrists ample protein with limited variability across biological replicates, factors that are important to establish technical reproducibility when novel workflows are being developed. The cardiovascular sciences have also influenced and benefited from developments in unbiased and targeted mass spectrometry (MS) approaches ( Figure 1B). Research pertaining to the heart and its metabolism, 4,5 extracellular matrix remodeling 6,7 posttranslational modification in vascular development. 8 lipoprotein content and metabolism, 9-11 and platelet function 12,13 have thrived as a result of the application of MS technologies. Recently, cardiovascular disease research has taken advantage of the readily accessible plasma pool for biomarker-and target-based studies. Tissues affected by heart failure, atherosclerosis and aortic aneurysms are in direct contact with the circulatory system, which increases the likelihood that proteins either causal or consequential to these pathologies can be detected in plasma. The use of mass spectrometry (MS)-dependent protein research is increasing in the cardiovascular sciences. A major reason for this is the versatility of and ability for MS technologies to accommodate a variety of biological questions such as those pertaining to basic research and clinical applications. In addition, mass spectrometers are becoming easier to operate, and require less expertise to run standard proteomics experiments. Nonetheless, despite the increasing interest in proteomics, many non-expert end users may not be as familiar with the variety of mass spectrometric tools and workflows available...

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Interference-Free Proteome Quantification with MS/MS-based Isobaric Isotopologue Detection

Cited by 24 publications

References 22 publications

Peptide Dimethylation: Fragmentation Control via Distancing the Dimethylamino Group

Peptide Dimethylation: Fragmentation Control via Distancing the Dimethylamino Group

NeuCode Labels for Relative Protein Quantification

Current Trends and Future Perspectives of State-of-the-Art Proteomics Technologies Applied to Cardiovascular Disease Research

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