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Intact Protein Characterization

Reputable Mentor II
Reputable Mentor II

Full characterization of a protein by mass spectrometry includes determination of the protein sequence, and identification and relative quantitation of protein isoforms, including identification and localization of one or multiple post-translational modifications (PTMs). Traditional analyses use a “bottom-up” approach, where proteins are digested into their peptide counterparts. However, complete sequence coverage is rarely attainable, and qualitative and quantitative information about protein isoforms, including those resulting from post-translational modifications, is usually lost.
Top-down protein characterization by mass spectrometry is an emerging technology that has several advantages over bottom up sequencing. Top-down analysis initially involves accurate measurement of molecular weight of intact protein followed by the fragmentation of the molecular ion in the gas phase. Top-down analysis facilitates direct observation of C- and N-termini for identification of truncations, preserves the relationship between modifications in any given isoform and allows quantitative differentiation between isoforms. High mass accuracy and high mass resolution are absolute requirements for this approach due to the complexity of MS/MS data.

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Workflow Overview for Intact Protein Characterization

Full characterization of intact proteins by top-down mass spectrometry is most commonly performed on proteins that have been enriched or purified. The enriched protein mixture is introduced into the mass spectrometer using either direct infusion or liquid chromatography coupled to an ESI source. Direct infusion provides more time for signal averaging and facilitates use of multiple fragmentation techniques. For more complex intact-protein mixtures, however, on- or off-line LC separation may be required to reduce precursor spectral complexity and minimize ion suppression. Users can choose a variety of fragmentation techniques for primary sequence determination. These include collisional activations and electron-capture based dissociations. Subsequent data analysis identifies and characterizes the protein sequence based both on accurate precursor mass and fragment masses from various dissociation experiments.


Sample Preparation

Sample Preparation Workflow for Intact Protein Characterization

Proper sample preparation is essential to the success of any top-down protein characterization experiment. For optimal characterization, proteins of interest are usually purified using methods such as immunoprecipitation. Removal of detergents and salts prior to MS analysis reduces both ion suppression and spectral interferences resulting in improved data quality. The easy-to-use Thermo Scientific Pierce Zeba Spin Desalting Columns and Detergent Removal Spin Columns can quickly clean up samples with minimal protein loss (1).

A comprehensive Mass Spectrometry Sample Preparation Handbook can be downloaded here.



1. Efficient removal of detergents from paroteins and peptides in a spin column format

Antharavally BS, Mallia KA, et al.
Anal Biochem. 2011 Sep 1;416(1):39-44.

related Products

Mass Spectrometry Sample Preparation Handbook

Zeba Spin Desalting Columns

Detergent Removal Spin Columns 

Mass Spectrometry

Mass Spectrometry Workflow for Intact Protein Characterization

Here are few tips for creating Orbitrap-based acquisition methods:

  • With electrospray ionization, there are typically many different charge states per intact protein. Fragmentation of different charge states may provide complementary sequence information. For example, although the low m/z ions may not be the most intense, these are often the best for ETD fragmentation due to the high charge density.


  • Proteins fragment differently depending on technique employed. In some cases CID fragmentation will offer more complete sequence coverage and in others ETD fragmentation will be optimal. Analyses with multiple, complementary fragmentation modes will improve the fragment ion coverage of the protein sequence, assisting in localization of sequence polymorphisms and post-translational modifications.


  • In addition to multiple fragmentation techniques, altering the parameters of a fragmentation experiment can also provide complementary sequence information. For example, ETD reaction times of 2-5 milliseconds will provide larger fragments at higher charge states, while longer reaction times of 20-50 milliseconds will result in smaller fragments and better coverage of termini.


  • Using multiple microscans averages multiple transients (1 microscan is equal to a single time transient) which are then used to generate a single high-resolution spectrum. This improves the signal-to-noise ratio of the resulting spectrum by the square root of the number of microscans averaged. Performing direct infusion experiments manually in Tune is recommended as one can use the average function for continuous transient averaging, while keeping the microscans settings to 1. For LC-based experiments microscans should be increased to 3-5, to ensure high-quality MS/MS spectra.


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Data Analysis

Data Analysis Workflow for Intact Protein Characterization

The intact mass of proteins can be calculated from their isotopically resolved MS spectra using Xtract algorithm within Thermo Scientific Xcalibur software and/or Thermo Scientific Protein Deconvolution software. If the protein spectra are not isotopically resolved, a specialized deconvolution algorithm called the ReSpect algorithm in Protein Deconvolution software version 2.0 can be applied.

Fully characterizing intact proteins at the MS/MS level comes with a variety of challenges. The fragmentation of large molecules, such as proteins, creates highly complex spectra due to the sheer number of fragments produced. Each is usually present at multiple m/z due to the different charge states. Also, a variety of modifications including post-translational modifications, cleavages, disulfide bonds, single-nucleotide polymorphisms, can occur anywhere within the sequence. Fragmentation data is used to accurately localize these changes.

These challenges can be met using the new Thermo Scientific ProsightPC software version 3.0 (1). It is the leading stand-alone software package for the identification of intact proteins and protein digests using high-resolution, accurate-mass MS and MS/MS data. It can process data generated using multiple fragmentation techniques including CID, HCD, and ETD. It is also the only proteomics software that allows the user to search their tandem MS data against proteome databases that incorporate biological annotations from UniProt, including PTMs, SNPs, and protein sequence isoforms. ProSightPC software also provides powerful search options including biomarker, delta M, and sequence tag; enabling detection of truncated and highly modified proteins.

Sample data results:


For more information on ProSightPC 3.0 software, please visit the Thermo Scientific Proteomics Software Portal.



1. Web and database software for identification of intact proteins using "top down" mass spectrometr...

Taylor GK, Kim YB, et al.
Anal Chem. 2003 Aug 15;75(16):4081-6.


Related Resources


Mapping intact protein isoforms in discovery mode using top-down proteomics

Tran JC, Zamdborg L, et al.
Nature. 2011 Oct 30;480(7376):254-8.
FanYu paper in Nature Biotech (2002) original ProsightPC paper

Identification and characterization of intact proteins in complex mixtures using online fragmentatio...

Accompanying Video Poster

Eliuk S, Kellie J, et al.
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Top-down Protein Sequencing and MS3 on a Hybrid Linear Quadrupole Ion Trap-Orbitrap Mass Spectromete...

Macek B, Waanders LF, et al.
Mol Cell Proteomics. 2006 May;5(5):949-58.

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Second T, Zabrouskov V, Makarov A.
Application Note 498

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Read more about ProSightPC software for precision proteomics

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Last update:
‎10-15-2021 02:12 AM
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