Versatile and multiplexed mass spectrometry-based absolute quantification with cell-free-synthesized internal standard peptides

Preparation of stable isotope-labeled internal standard peptides is crucial for mass spectrometry (MS)-based targeted proteomics. Herein, we developed versatile and multiplexed absolute protein quantification method using MS. A previously developed method based on the cell-free peptide synthesis system, termed MS-based quantification by isotope-labeled cell-free products (MS-QBiC), was improved for multiple peptide synthesis in one-pot reaction. We pluralized the quantification tags used for the quantification of synthesized peptides and thus, made it possible to use cell-free synthesized isotope-labeled peptides as mixtures for the absolute quantification. The improved multiplexed MS-QBiC method was proved to be applied to clarify ribosomal proteins stoichiometry in the ribosomal subunit, one of the largest cellular complexes. The study demonstrates that the developed method enables the preparation of several dozens and even several hundreds of internal standard peptides within a few days for quantification of multiple proteins with only a single-run of MS analysis.

Download Full-text

Quantitative Performance of Internal Standard Platforms for Absolute Protein Quantification Using Multiple Reaction Monitoring-Mass Spectrometry

Analytical Chemistry ◽

10.1021/acs.analchem.5b00331 ◽

2015 ◽

Vol 87 (8) ◽

pp. 4429-4435 ◽

Cited By ~ 44

Author(s):

Kerry Bauer Scott ◽

Illarion V. Turko ◽

Karen W. Phinney

Keyword(s):

Mass Spectrometry ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

Protein Quantification ◽

Reaction Monitoring ◽

Quantitative Performance

Download Full-text

Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs

Nucleic Acids Research ◽

10.1093/nar/gku661 ◽

2014 ◽

Vol 42 (15) ◽

pp. 9880-9891 ◽

Cited By ~ 41

Author(s):

Arne H. Smits ◽

Rik G.H. Lindeboom ◽

Matteo Perino ◽

Simon J. van Heeringen ◽

Gert Jan C. Veenstra ◽

...

Keyword(s):

Single Cell ◽

Ribosomal Proteins ◽

Single Cells ◽

Transcript Level ◽

Absolute Quantification ◽

Protein Quantification ◽

Protein Levels ◽

Recent Developments ◽

Basal Transcription Factors ◽

Cell Proteome

Abstract While recent developments in genomic sequencing technology have enabled comprehensive transcriptome analyses of single cells, single cell proteomics has thus far been restricted to targeted studies. Here, we perform global absolute protein quantification of fertilized Xenopus laevis eggs using mass spectrometry-based proteomics, quantifying over 5800 proteins in the largest single cell proteome characterized to date. Absolute protein amounts in single eggs are highly consistent, thus indicating a tight regulation of global protein abundance. Protein copy numbers in single eggs range from tens of thousands to ten trillion copies per cell. Comparison between the single-cell proteome and transcriptome reveal poor expression correlation. Finally, we identify 439 proteins that significantly change in abundance during early embryogenesis. Downregulated proteins include ribosomal proteins and upregulated proteins include basal transcription factors, among others. Many of these proteins do not show regulation at the transcript level. Altogether, our data reveal that the transcriptome is a poor indicator of the proteome and that protein levels are tightly controlled in X. laevis eggs.

Download Full-text

Versatile and multiplexed mass spectrometry-based absolute quantification with cell-free-synthesized internal standard peptides

Journal of Proteomics ◽

10.1016/j.jprot.2021.104393 ◽

2021 ◽

pp. 104393

Author(s):

Keiko Masuda ◽

Keiko Kasahara ◽

Ryohei Narumi ◽

Masaru Shimojo ◽

Yoshihiro Shimizu

Keyword(s):

Mass Spectrometry ◽

Internal Standard ◽

Absolute Quantification

Download Full-text

Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry–Based Assays

Clinical Chemistry ◽

10.1373/clinchem.2015.250563 ◽

2016 ◽

Vol 62 (1) ◽

pp. 48-69 ◽

Cited By ~ 95

Author(s):

Andrew N Hoofnagle ◽

Jeffrey R Whiteaker ◽

Steven A Carr ◽

Eric Kuhn ◽

Tao Liu ◽

...

Keyword(s):

Mass Spectrometry ◽

Clinical Care ◽

Internal Standard ◽

Mass Spectrometric ◽

Protein Quantification ◽

Analytical Sensitivity ◽

Central Importance ◽

Protein Mass ◽

Clinical Laboratories ◽

Protein Assays

Abstract BACKGROUND For many years, basic and clinical researchers have taken advantage of the analytical sensitivity and specificity afforded by mass spectrometry in the measurement of proteins. Clinical laboratories are now beginning to deploy these work flows as well. For assays that use proteolysis to generate peptides for protein quantification and characterization, synthetic stable isotope–labeled internal standard peptides are of central importance. No general recommendations are currently available surrounding the use of peptides in protein mass spectrometric assays. CONTENT The Clinical Proteomic Tumor Analysis Consortium of the National Cancer Institute has collaborated with clinical laboratorians, peptide manufacturers, metrologists, representatives of the pharmaceutical industry, and other professionals to develop a consensus set of recommendations for peptide procurement, characterization, storage, and handling, as well as approaches to the interpretation of the data generated by mass spectrometric protein assays. Additionally, the importance of carefully characterized reference materials—in particular, peptide standards for the improved concordance of amino acid analysis methods across the industry—is highlighted. The alignment of practices around the use of peptides and the transparency of sample preparation protocols should allow for the harmonization of peptide and protein quantification in research and clinical care.

Download Full-text

A comparative study of synthetic winged peptides for absolute protein quantification

Scientific Reports ◽

10.1038/s41598-021-90087-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Eliska Benesova ◽

Veronika Vidova ◽

Zdenek Spacil

Keyword(s):

Amino Acid ◽

Water Solubility ◽

Internal Standard ◽

Absolute Quantification ◽

Protein Quantification ◽

Amino Acid Residues ◽

C Terminus ◽

Enzymatic Proteolysis ◽

Quantitative Performance ◽

C And N

AbstractA proper internal standard choice is critical for accurate, precise, and reproducible mass spectrometry-based proteomics assays. Synthetic isotopically labeled (SIL) proteins are currently considered the gold standard. However, they are costly and challenging to obtain. An alternative approach uses SIL peptides or SIL "winged" peptides extended at C- or/and N-terminus with an amino acid sequence or a tag cleaved during enzymatic proteolysis. However, a consensus on the design of a winged peptide for absolute quantification is missing. In this study, we used human serum albumin as a model system to compare the quantitative performance of reference SIL protein with four different designs of SIL winged peptides: (i) commercially available SIL peptides with a proprietary trypsin cleavable tag at C-terminus, (ii) SIL peptides extended with five amino acid residues at C-terminus, (iii) SIL peptides extended with three and (iv) with five amino acid residues at both C- and N-termini. Our results demonstrate properties of various SIL extended peptides designs, e.g., water solubility and efficiency of trypsin enzymatic cleavage with primary influence on quantitative performance. SIL winged peptides extended with three amino acids at both C- and N-termini demonstrated optimal quantitative performance, equivalent to the SIL protein.

Download Full-text

Structural organization of escherichia coli ribosomes as determined by immuno electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081693 ◽

1978 ◽

Vol 36 (2) ◽

pp. 166-167 ◽

Cited By ~ 1

Author(s):

G. Stöffler ◽

R.W. Bald ◽

J. Dieckhoff ◽

H. Eckhard ◽

R. Lührmann ◽

...

Keyword(s):

Electron Microscopy ◽

Escherichia Coli ◽

Ribosomal Proteins ◽

Structural Organization ◽

Three Dimensional ◽

Ribosomal Subunit ◽

Spatial Arrangement ◽

Small Subunit ◽

Antibody Binding ◽

Immuno Electron Microscopy

A central step towards an understanding of the structure and function of the Escherichia coli ribosome, a large multicomponent assembly, is the elucidation of the spatial arrangement of its 54 proteins and its three rRNA molecules. The structural organization of ribosomal components has been investigated by a number of experimental approaches. Specific antibodies directed against each of the 54 ribosomal proteins of Escherichia coli have been performed to examine antibody-subunit complexes by electron microscopy. The position of the bound antibody, specific for a particular protein, can be determined; it indicates the location of the corresponding protein on the ribosomal surface.The three-dimensional distribution of each of the 21 small subunit proteins on the ribosomal surface has been determined by immuno electron microscopy: the 21 proteins have been found exposed with altogether 43 antibody binding sites. Each one of 12 proteins showed antibody binding at remote positions on the subunit surface, indicating highly extended conformations of the proteins concerned within the 30S ribosomal subunit; the remaining proteins are, however, not necessarily globular in shape (Fig. 1).

Download Full-text

Protein-induced conformational changes in 16S rRNA during assembly of the Escherichia Coli 30S ribosomal subunit: A STEM study

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100128833 ◽

1987 ◽

Vol 45 ◽

pp. 910-911

Author(s):

M. Boublik ◽

V. Mandiyan ◽

J.F. Hainfeld ◽

J.S. Wall

Keyword(s):

16S Rrna ◽

Conformational Changes ◽

Ribosomal Proteins ◽

Structural Changes ◽

Ribosomal Subunit ◽

Compact Structure ◽

E Coli ◽

Freeze Dried ◽

16S Rna ◽

30S Subunit

The aim of this study is to understand the mechanism of 16S rRNA folding into the compact structure of the small 30S subunit of E. coli ribosome. The assembly of the 30S E. coli ribosomal subunit is a sequence of specific interactions of 16S rRNA with 21 ribosomal proteins (S1-S21). Using dedicated high resolution STEM we have monitored structural changes induced in 16S rRNA by the proteins S4, S8, S15 and S20 which are involved in the initial steps of 30S subunit assembly. S4 is the first protein to bind directly and stoichiometrically to 16S rRNA. Direct binding also occurs individually between 16S RNA and S8 and S15. However, binding of S20 requires the presence of S4 and S8. The RNA-protein complexes are prepared by the standard reconstitution procedure, dialyzed against 60 mM KCl, 2 mM Mg(OAc)2, 10 mM-Hepes-KOH pH 7.5 (Buffer A), freeze-dried and observed unstained in dark field at -160°.

Download Full-text