Amine-reactive Neutron-encoded Labels for Highly Plexed Proteomic Quantitation

Abstract

We describe a novel amine-reactive chemical label that exploits differential neutron-binding energy between 13 C and 15 N isotopes. These neutron-encoded (NeuCode) chemical labels enable up to 12-plex MS1-based protein quantification. Each structurally identical, but isotopically unique, tag is encoded with a 12.6-mDa mass differencerelative to its nearest neighbor-so that peptides bearing these NeuCode signatures do not increase spectral complexity and are detected only upon analysis with very high mass-resolving powers. We demonstrate that the method provides quantitative performance that is comparable to both metabolic labeling and isobaric tagging while combining the benefits of both strategies. Finally, we employ the tags to characterize the proteome of Saccharomyces cerevisiae during the diauxic shift, a metabolic transition from fermentation to aerobic respiration. Proteome quantification is an increasingly essential component of modern biology and translational medicine (1, 2). Whether targeted or global, stable isotope incorporation with mass spectrometry (MS) analysis is a core technique for protein abundance measurements. There are numerous approaches that can be used to introduce stable isotopes into peptides, the most frequently used being stable isotope labeling with amino acids in cell culture (SILAC) 1 and isobaric tagging (tandem mass tags or isobaric tags for relative and absolute quantitation) (3-7). Both of these methods incorporate heavy isotopes to increase mass by at least 1 Da. SILAC is the quantification gold standard for global proteomic analysis. However, the SILAC approach is not easily adapted for tissue sample analysis; SILAC mouse labeling, for example, requires feeding mice a specialized diet for multiple generations (8). Tissue samples can be analyzed if they are mixed with SILAC cell-culture-based labeled standards, but this strategy does not permit multiplexing (9). Isobaric labels, in contrast, are conjugated to the primary amines of peptides following proteolytic digestion and thus have the advantage of being completely compatible with samples from virtually any source (10, 11). That said, isobaric tagging suffers from dynamic range suppression caused by co-isolation of precursor peptides (12). Multiple studies have revealed that this problem greatly erodes quantitative accuracy-for example, 10-fold changes often are detected as much smaller ϳ4-fold changes (13,14). Here we propose a new approach to protein quantification, one that achieves tissue-compatible 4-plexed MS1-based quantification without increasing spectral complexity.Recently, we described the use of mass defects to expand SILAC quantification from 3-plex to 12-plex and beyond (i.e., NeuCode SILAC) (15). NeuCode SILAC exploits the subtle mass differences that exist in atoms as a result of the varying energies of nuclear binding in common stable isotopes (e.g., carbon, nitrogen, hydrogen, and oxygen) by using the extremely high resolving power of modern Fourier transform mass spectrometer systems (16,17). For ex...

Comprehensive Temporal Protein Dynamics during the Diauxic Shift in Saccharomyces cerevisiae

“…For example, using multiple reaction monitoring (MRM) to profile the diauxic shift has offered temporal coverage of central carbon metabolism, but at a depth of less than 100 proteins. Although not comprehensive, a shotgun approach employing four-plex labeling by aminereactive neutron-encoded labels has allowed more proteins to be quantified (1543) but with lower coverage of carbon metabolism and few time-points to delineate temporal patterns (21). More recently, six-plex TMT achieving comprehensive levels of protein quantifications of either log phase or stationary phase (6).…”

What Can Proteomics Tell Us About Platelets?

“…(1) Currently multiplexing is limited to only 3 samples and, as for all nonisobaric labels, 2-plexing will double and 3-plexing will triple the complexity of the sample. (2) Incorporation of deuterium causes retention time shifts between light/medium/ heavy forms of the peptide during reversed phase chromatography and thus can complicate quantification by liquid chromatography-MS. 65 Hebert et al 66,67 recently introduced a novel strategy for neutron-encoded labeling, which will increase the multiplexing capabilities of the aforementioned labeling strategies, but requires mass spectrometers with a resolution power of ≈500 000 and above, currently supported by only few instruments in conjunction with reasonable sensitivity and scanning speed. (6) with subsequent multiplexing and analysis of pooled samples can be applied.…”

Neuroproteomics — LC-MS Quantitative Approaches

“…This new approach is named neutron encoding (NeuCode) SILAC, where peptide identifications are generated using the MS1 scans collected at 30,000 resolving power, where the same peptide with multiple labels will appear as a single peak in the spectra, whereas to obtain the quantitative information a higher resolution (480,000) MS1 scan is used, where the isotopologues can be resolved and the quantitative information extracted as for normal SILAC (with a mass shift of 36 mDa instead of 4 or 8 Da) [62]. This approach has the advantage of decreasing redundant acquisition of fragment spectra for the same precursor ion (as in classical SILAC), and because the quantitative information is acquired at the MS1 level, it is not dependent on peptides selected for MS/MS and is not subjected to dynamic range compression caused by co-isolation of precursor ions (as in isobaric labeling, see below) [62,63].…”

“…In these first reports, the authors claim that the NeuCode approach may be used for 12-plexing by using 3-plex SILAC, each one combined with 4 isotopologues, resulting in four distinct peaks in a high-resolution spectra [62,63], although it has already been used for 6-and 18-plex in yeast cells proteome [64]. This approach has already been used in other applications, such as C-terminal product ion annotation, based on the fact that all the y-ion in the fragment spectra will appear as doublets [65,66], or in top-down proteomics (analysis of the intact proteins instead of peptides resulting from protein digestion) [67].…”

“…As for SILAC, very recently, the NeuCode strategy described above has been applied to chemical labeling with the development of an amine-reactive mass tag that takes advantage of the differential neutron-binding energy between 13 C and 15 N isotopes that enables up to 12-plex MS1-based protein quantification [63]. Another NeuCode approach proposed is to use carbamylation of amine groups via urea isotopologues for protein/peptide labeling, and therefore relative quantification [85].…”