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While more complex experiments such as relative and absolute quantification of proteins have become increasingly common, bottom-up protein identification continues to be a mainstay of proteomics. Its principal objective is to identify as many of the protein components of a biological sample as possible. Following digestion, peptides are identified by LC/MS. The resulting sequence data is used to determine the original protein components of the sample. With advances in mass resolution, mass accuracy, fragmentation technology and speed, bottom-up analysis can identify more proteins in more-complex samples than ever. Moreover, the techniques and principles involved are generally foundational for more complex experiments. Here we describe the basic bottom-up experiment, with attention to performance optimization for various sample types.

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Overview

Workflow for Bottom-Up Proteomics

 

Bottom-up proteomics serves as the basis for much of the protein research undertaken in mass spectrometry laboratories today. The term ”bottom-up” implies that information about the constituent proteins of a biological sample are reconstructed from individually identified fragment peptides. To facilitate bottom-up MS analysis, proteins are subjected to proteolytic digestion, typically using trypsin. The resulting peptides are usually separated using one or more dimensions of liquid chromatography. The LC eluent is interfaced to a mass spectrometer using electrospray ionization and the fragment peptides are analyzed by MS.

 


Literature Highlights

Advancing cell biology through Proteomics in Space and Time (PROSPECTS)

Lamond AI, Uhlen M, et al.
Mol Cell Proteomics. 2012 Mar;11(3):O112.017731.


 


Workflow Overview for Bottom-Up Proteomics

 

Bottom-up proteomics serves as the basis for much of the protein research undertaken in mass spectrometry laboratories today. The term ”bottom-up” implies that information about the constituent proteins of a biological sample are reconstructed from individually identified fragment peptides. To facilitate bottom-up MS analysis, proteins are subjected to proteolytic digestion, typically using trypsin. The resulting peptides are usually separated using one or more dimensions of liquid chromatography. The LC eluent is interfaced to a mass spectrometer using electrospray ionization and the fragment peptides are analyzed by MS.
 


 

Workflows_BottomUpProt-2.jpg


 


 

Literature Highlight

 

Advancing cell biology through Proteomics in Space and Time (PROSPECTS)

Lamond AI, Uhlen M, et al.
Mol Cell Proteomics. 2012 Mar;11(3):O112.017731.
 

Pushing the Limits of Bottom-Up Proteomics with State-Of-The-Art Capillary UHPLC and Orbitrap Mass S...

Lopez-Ferrer D, Blank M , Meding, et al.
Application Note 639


 

Mass Spectrometry

Mass Spectrometry Workflow for Bottom-up Proteomics

 

In recent years, electrospray (ESI)-based MS/MS analysis coupled to online reverse-phase LC separation has become the default choice for analysis of peptides. This is due primarily to the excellent behavior of tryptic peptides under ESI conditions and to the fact that experimenters have sought to analyze ever more complex peptide mixtures, up to and including digestions of entire tissues or organisms. Current nanoscale HPLC methods typically provide a peak capacity on the order of 400-700 chromatographic peaks across an analytical gradient, meaning that the momentary complexity of the sample, as it is being introduced into the mass spectrometer, can be reduced. Many thousands of individual peptides can be analyzed in a single analytical run using modern LC-MS/MS techniques.

 
The optimal set of MS instrument method parameters will vary greatly depending on the instrument type and sample complexity. There is always a relationship between the overall complexity of the sample and the amount of time the instrument can spend analyzing any specific ion species. For highly complex samples, it is generally advantageous to set relatively low limits on the maximum time the instrument can spend on any target, in order to maximize the number of targets analyzed. For less complex samples, the instrument can be allowed to spend more time on each precursor, for example by setting higher limits on the amount of time an ion trap may be filled (max injection time) or by averaging replicate spectra to improve signal-to-noise ratios (increasing microscans).
 
Using multiple fragmentation modes can also benefit bottom-up peptide analysis. ETD data has been shown to yield complementary spectral information to CID. EThcD has shown to be beneficial for post-translationally modified peptides.1-3 More peptides can be identified when these fragmentation modes are used in a data-dependent decision tree, selecting the fragmentation method based on the m/z and charge state of the precursor ion.4 HCD can further complement a complex analysis by identifying peptides not identified by ETD or CID. 5
 
The number of precursor ions analyzed per cycle (TopN) is dependent on acquisition scan rate. Faster instruments benefit from higher N. Optimal N also depends on complexity of the sample, amount loaded, and chromatography. For sample loads of 1 µg or more, Top20 is a common starting point for Q Exactive HF instrument while TopS is recommended for Orbitrap Fusion and Orbitrap Fusion Lumos instruments.
 
Using higher resolution for full-scan MS allows the detection of more precursors, particularly at low sample levels.  Resolution in excess of 120,000 FWHM at m/z 400 was found to be optimal when analyzing complex samples using Orbitrap Fusion Lumos system.

References

 

1. Unambiguous phosphosite localization using electron-transfer/higher-energy collision dissociation...

Frese CK, Zhou H, Taus T, Altelaar AF, Mechtler K, Heck AJ, Mohammed S.
J Proteome Res 2013 12(3), 1520-5.


2. Toward full peptide sequence coverage by dual fragmentation combining electron-transfer and highe...

Frese CK, Altelaar AF, van den Toorn H, Nolting D, Griep-Raming J, Heck AJ, Mohammed S
Anal Chem 2012 84(22), 9668-73.
 

3. Extended O-GlcNAc on HLA Class-I-Bound Peptides

Marino F, Bern M1, Mommen GP2, Leney AC, van Gaans-van den Brink JA3, Bonvin AM, Becker C1, van Els CA3, Heck AJ
J Am Chem Soc 2015 37(34), 10922-5.


4.
Decision tree-driven tandem mass spectrometry for shotgun proteomics

Swaney DL, McAlister GC, Coon JJ.
Nat Methods. 2008 Nov;5(11):959-64.

 

5. A Dual Pressure Linear Ion Trap Orbitrap Instrument with Very High Sequencing Speed

Olsen JV, Schwartz JC, et al.
Mol Cell Proteomics. 2009 Dec;8(12):2759-69.

ADDITIONAL RESOURCES

 

System-wide perturbation analysis with nearly complete coverage of the yeast proteome by single-shot...

Nagaraj N, Kulak NA, et al.
Mol Cell Proteomics. 2012 Mar;11(3):M111.013722.
 

Performance of a linear ion trap-Orbitrap hybrid for peptide analysis.

Yates JR, Cociorva D, et al.
Anal Chem. 2006 Jan 15;78(2):493-500.
 

A Quadrupole-Orbitrap Hybrid Mass Spectrometer Offers Highest Benchtop Performance for In-Depth Anal...

Hao Z, Zhang Y, et al.
Application Note 552
 

The Q Exactive HF, a Benchtop Mass Spectrometer with a Pre-filter, High-performance Quadrupole and a...

Scheltema RA, Hauschild J, Lange O, Hornburg D, et al.
Mol Cell Proteomics 2014, 13(12), 3698-3708.

 
Rapid and Deep Proteomes by Faster Sequencing on a Benchtop Quadrupole Ultra-High-Field Orbitrap Ma...

Kelstrup CD, Jersie-Christensen R.R, Batth TS, et al.
J Proteome Res 2014, 13 (12), 6187-6195.


The one hour yeast proteome

Hebert AS, Richards AL, Bailey DJ, Ulbrich A, Coughlin EE, Westphall MS, Coon JJ.
Mol Cell Proteomics 2014 13(1), 339-47.
 

Pushing the Limits of Bottom-Up Proteomics with State-Of-The-Art Capillary UHPLC and Orbitrap Mass S...

Lopez-Ferrer D, Blank M , Meding, et al.
Application Note 639

  

Standardized Workflows for Near Complete Proteome Identification

  Jiang X, Xu S, Wang Z, et al.
Scientific Poster

Data Analysis

Data Analysis Workflow for Bottom-Up Proteomics

 

There are a variety of algorithms for the interpretation of peptide fragmentation data. The most commonly employed algorithms, such as those used by SEQUEST®1, Mascot™ and Byonic™, attempt to determine the identity of a peptide by comparing the observed fragmentation pattern to theoretical fragmentation patterns derived from protein sequence databases and heuristic fragmentation rules. The observed mass of the intact precursor ion is used to constrain the set of theoretical peptides that are considered within a tolerance range based on the accuracy of the measurement. Instruments that provide high mass accuracy precursor measurements allow greatly improved search times and improved confidence in peptide identifications2, especially for modified peptides.3 High mass accuracy is also beneficial in MSn (fragmentation) spectra, though somewhat less consequential than in MS1 because the information identifying the peptide is spread across multiple fragment ions, and greater uncertainty in mass measurement can usually be tolerated.
 

Thermo Scientific Proteome Discoverer software has a comprehensive set of tools for the data mining of CID, HCD, ETD and EThcD spectra using SEQUEST Mascot and Byonic, including mixed raw files containing spectra from multiple fragmentation modes in a single run. For detailed step-by-step information about data analysis using Proteome Discoverer software and to download a free 60-day demonstration version of the latest Proteome Discoverer software, please visit the Thermo Scientific Proteomics Software Portal.


An emerging analytical approach involves the use of spectral libraries. These are sets of previously acquired fragmentation spectra, usually annotated and filtered for quality. Experimental spectra are compared to library spectra directly, rather than to theoretical spectra, to provide identification. This has the advantage of using information about the actual fragmentation behavior of a given peptide, rather than generalized fragmentation rules, thus improving the accuracy of the pattern matching.4 Spectral library searches can also be much faster than theoretical database searches, due to a much smaller search space. The principal liability of this approach is that new identifications cannot be made; spectra not already in the database will not be identified.

References

 

1. Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the prote...

Yates JR 3rd, Eng JK, et al.
 

2. Precision proteomics: The case for high resolution and high mass accuracy

Mann M, Kelleher NL.
Proc Natl Acad Sci U S A. 2008 Nov 25;105(47):18132-8.
 

3. Optimization and use of peptide mass measurement accuracy in shotgun proteomics

Haas W, Faherty BK, et al.
Mol Cell Proteomics. 2006 Jul;5(7):1326-37.
 

4. Using BiblioSpec for creating and searching tandem MS peptide libraries

Frewen B, MacCoss MJ.
Curr Protoc Bioinformatics. 2007 Dec;Chapter 13:Unit 13.7.

 Additional Resources

 

Comparison and Combination of Search Engines to Discover and Characterize Identifications and PTM Si...

Jiang X, Horn D, Blank M, et al.
Poster Note



LINKS


Read more about Proteome Discoverer mass informatics platform for protein scientists
LEARN MORE>

To download Proteome Discoverer software, please visit the Thermo Scientific Proteomics Software Portal.

Sample Preparation

Sample Prep Workflow for Bottom-up Proteomics


Given the very general applicability of bottom-up proteomics, there are a multitude of sample preparation techniques. Which technique is most appropriate generally depends on the source, type, and complexity of the sample. Samples can derive from a wide variety of biological sources and span a wide range of complexities.  At one extreme, a researcher may seek to analyze a single purified protein.1 At the other extreme, the objective may be to catalog thousands of proteins derived from a cell or tissue sample.2-4  Experiments of intermediate complexity can involve analysis of purified protein complexes5,  fractionated protein mixtures such as an excised gel band6, or a chromatographic fraction.7
 
Regardless of the origin of the protein(s) to be analyzed, once the sample is prepared, the next step in bottom-up analysis is the generation of peptides that are suitable for MS analysis. Peptide preparation involves reduction and alkylation of cysteines, digestion of the sample into peptides, desalting and concentration of the peptides.
 
Trypsin is by far the most commonly used digestion enzyme. It cuts C-terminally to arginine and lysine residues, when not blocked by an adjacent proline residue. This has the advantage of generating peptides that are of moderate size due to the natural abundance rates of these amino acids and that tend to carry two or three positive charges when ionized by electrospray. Tryptic peptides are generally optimal for MS/MS analysis via collision induced dissociation as their charge state and length provide ready fragmentation yielding information-rich, but not overly complex spectra. Alternative enzymes, such as GluC, AspN, and LysC, are sometimes used when larger peptides are required or when a region of interest within a given protein will not yield a suitable tryptic peptide.8

REferences

 

1. Quantification of post-translationally modified peptides of bovine alpha-crystallin using tandem ma...

Viner RI, Zhang T, et al.
J Proteomics. 2009 Jul 21;72(5):874-85.
 

2. System-wide perturbation analysis with nearly complete coverage of the yeast proteome by single-sho...

Nagaraj N, Kulak NA, et al.
Mol Cell Proteomics. 2012 Mar;11(3):M111.013722.
 

3. Evaluation of HCD- and CID-type fragmentation within their respective detection platforms for murine...

Jedrychowski MP, Huttlin EL, et al.
Mol Cell Proteomics. 2011 Dec;10(12):M111.009910.
 

4. Decision tree-driven tandem mass spectrometry for shotgun proteomics

Swaney DL, McAlister GC, Coon JJ.
Nat Methods. 2008 Nov;5(11):959-64.
 

5. Topographic studies of the GroEL-GroES chaperonin complex by chemical cross-linking using diformyl e...

Trnka MJ, Burlingame AL.
Mol Cell Proteomics. 2010 Oct;9(10):2306-17.


6. Phosphoproteome Analysis of Fission Yeast

Wilson-Grady JT, Villén J, Gygi SP.
J Proteome Res. 2008 Mar;7(3):1088-97.
 

7. Proteomics by mass spectrometry: approaches, advances, and applications

Yates JR, Ruse CI, Nakorchevsky A.
Annu Rev Biomed Eng. 2009;11:49-79.
    
  

8. Mass spectrometry identifies and quantifies 74 unique histone H4 isoforms in differentiating human e...

Phanstiel D, Brumbaugh J, et al.

Proc Natl Acad Sci U S A. 2008 Mar 18;105(11):4093-8.

ADDITIONAL RESOURCES

 

Mass Spec Analysis Technical Handbooks

Information on new products and any of our technical handbooks to help you improve your mass spectrometry experiments can be accessed here.



Protein Digestion for Mass Spectrometry – Available Tools

 

 

Grant Central

Grant Central Resources for Bottom-up Proteomics


Every research idea matters. At Thermo Fisher Scientific, we are dedicated to helping you advance your research, and that includes becoming your scientific partner in supporting your grant application efforts. Our latest grant writing resources are listed below.

Need supporting information for your grant proposal or have a grant writing related question? Visit Grant Central or Contact Us.
 


GENERAL Resources

 

Top 5 reasons to upgrade from a Thermo Scientific™ Hybrid Orbitrap™ to a Thermo Scientific™ Tribrid™...
Grant Application Resource
 

Technical Resources


Low Attomole Limit of Quantification on an Orbitrap Fusion Lumos Tribrid Mass Spectrometer
Poster Note
 

A Universal Method for Peptide Identification
Poster Note

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Last update:
‎08-05-2021 08:30 PM
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