The Identity^E High Definition Proteomics System

The Identity^E High Definition Proteomics System sets high standards for rigorous protein identification. Designed for authoritative and comprehensive qualitative analysis, the Waters solution enables proteomics researchers to reference chromatographic retention time, precursor and product ion masses, structural significance, biological context, and plausible detectability of multiple peptides to confidently identify a protein.

The Identity^E System is designed exclusively for qualitative analysis of complex protein digests, taking advantage of the superior resolution of UPLC separations and the novel approach of MS^E, providing outstanding chromatographic results and simultaneous precursor and product ion information.

The system includes comprehensive peptide ion accounting informatics. This software helps you visualize and identify peptides and proteins with a multi-layered physicochemical model of protein primary structure, for generation of the highest quality MRM transitions for tandem quadrupole biomarker verification and validation.

The Identity^E System provides complete and contextually-based information for confident protein identification – that can lead to more promising biological and medical research. This includes laboratories looking for improved reproducibility and reliability for qualitative protein identification, such as:

• University biology/biochemistry departments
• Core proteomic laboratories
• Cancer research facilities
• Protein function/interaction laboratories
• Biotechnology companies

Waters Identity^E High Definition Proteomics System configuration:

• nanoACQUITY UPLC System
• Waters QTof or Ion Mobility Mass Spectrometry systems
• MS^E data acquisition and dynamic mass resolution
• Comprehensive peptide ion accounting informatics for high protein sequence coverage, enabling you to identify the most proteins with the lowest error rate.
• ProteinLynx GlobalSERVER (PLGS) software
• Training and support, including online courses

The Identity^E High Definition Proteomics System combines UPLC/MS^E data with a comprehensive peptide ion accounting informatics solution to catalog complex protein digest mixtures.

The system features:

• Kinetically-tuned protein digestion protocol to marginalize missed cleavages
• Direct nanoflow UPLC separations with rugged sub-2-μm Bridged Ethyl Hybrid (BEH) particle columns that deliver unprecedented peak capacity and retention time repeatability
• UPLC peak capacity and robustness accentuates the specificity of the Identity^E System’s retention time prediction model, allowing the amino acid composition of peptides, identified from matching MS^E spectra, to be corroborated
• High resolution mass spectrometry enables peptide detection using dynamic mass resolution and bandwidth to ensure consistent over-sampling of complex protein digests
• Identity^E is extendable through the Expression^E System to label-free relative and absolute quantification studies for protein biomarker discovery

The Identity^E system dramatically increases sequence coverage in this study of a digested cytosolic fraction from an E.coli lysate (three technical replicas).

Data Acquisition by Alternating Spectra Energy

UPLC/MS^E is a unique, patented technology that has an extremely high duty cycle, allowing the acquisition of high quality accurate mass data in a simple, unbiased manner.

UPLC/MS^E acquires data with two rapidly alternating MS functions. The first contains exclusively low-energy mass spectra, the second is composed of mass spectra acquired at elevated collision energy (designated as MS^E). The resulting data set contains a comprehensive time-resolved record of all detectible precursor and product ions.

Precursor and product ions are associated by both retention time alignment and peak shape, enabling even chimeric peptides and overlapping ion clusters to be successfully handled. The resulting time-aligned precursor and product ion mass lists are used for peptide identification with the Identity^E System’s peptide ion informatics.

Peptide Ion Informatics

This bioinformatics software visualizes peptides and proteins with a new multi-layered physicochemical model of protein primary structure. Peptide ion accounting informatics adapts to each new UPLC/MS^E data set using a pre-assessment survey. 250 identified peptides, per run, are used to fine-tune many of the 14 models used for both peptide and protein authentication.

After pre-assessment, an iterative database search strategy is initiated, identifying the most abundant protein within a sample by matching peptides, modified peptides, and retention times. An estimate of the absolute concentration of each protein is made by reference to an internal (protein) standard, allowing the fractions of all theoretical tryptic peptides that are plausibly detectable to be predicted. Proteins identified with fewer than the predicted number of peptides are sequence mapped for evidence of C/N-terminal processing.

When the most abundant protein is conclusively identified, all assigned peptide ions are subtracted from the working image of the data.

Multi-layered Physicochemical Model

The Identity^E System features Waters peptide ion accounting informatics. This technology visualizes peptides and proteins with a multi-layered physicochemical model of protein primary structure, developed from a post-genomic understanding of protein characteristics – an advantage unavailable in any other protein identification tool and unique to the Identity^E system.

Pre-assessment survey

Peptide ion accounting informatics adapts to each new UPLC/MS^E data set utilizing a pre-assessment survey. 250 unambiguously identified (abundant) peptides, per run, are used to fine-tune many of the 14 models used for both peptide and protein authentication. Automatic adjustment of these models on an injection-by-injection basis provides the means for the software to handle changes in chromatographic conditions (buffers and columns), enzymatic specificity, and sample complexity.

Fourteen different physicochemical attributes are automatically considered to maximize peptide identification confidence. Other conventional protein identification systems utilize only a small number of these characteristics.

Iterative Protein Identification

When the most abundant protein is conclusively identified, all assigned peptide ions are subtracted from the working image of the data. The process is then repeated for the next most abundant protein, as many times as is necessary to account for as many ions as possible within the UPLC/MS^E data set. As more proteins are identified, and their associated peptide ions are removed, the working image of the data becomes less complex – revealing proteins of decreasing abundance for identification.

Utilizing the 14 physicochemical models of the peptide ion accounting informatics, proteins are identified in order of their abundance, thus increasing the selectivity, specificity, and sensitivity of each iterative protein identification. The above data illustrates that proteins are being identified and their respective ions depleted by their abundance.

More Peptides per Protein

The Identity^E System identifies more peptides per protein, providing both higher sequence coverage and rigor to the protein ID. Over 95% (389) of the proteins were identified with greater than three peptides to match, compared to the LTQ Orbitrap and the LTQ with only 65% (244) and 33% (88), respectively.