Simplification of complex peptide mixtures for proteomic analysis: Reversible biotinylation of cysteinyl peptides

Spahr, Chris; Susín, Santos A.; Bures, Edward J.; Robinson, John H.; Davis, Michael T.; McGinley, Michael D.; Kroemer, Guido; Patterson, Scott D.

doi:10.1002/(sici)1522-2683(20000501)21:9<1635::aid-elps1635>3.0.co;2-1

Cited by 91 publications

(35 citation statements)

References 20 publications

(22 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To improve the odds of obtaining some peptide sequence from all of the proteins comprising the 39 S subunit, three types of analyses were used: 1) replicate nanoscale capillary LC/MS/MS of both trypsin and Lys-C digests; 2) Cys-affinity labeling/fractionation (18); and 3) on-line multidimensional SCX-RP LC/LC/MS/MS (16). The most straightforward method for improving proteome coverage of 39 S proteins was found to be replicate analysis of identical digests of the subunit.…”

Section: Resultsmentioning

confidence: 99%

“…This data illustrates that the number of 39 S proteolytic peptides presented to the mass spectrometer for interrogation in an LC/MS/MS experiment exceeds the sequencing capacity of the instrument. Because chance (to some extent) plays a role in which peptide ions the instrument interrogates in each analysis (18), simply repeating the analysis results in a substantial number of new peptides interrogated in each experiment.…”

Section: Resultsmentioning

confidence: 99%

“…2) was the only peptide from MRP-L55 observed in any of our analyses. Another LC/LC approach taken was the use of Cys affinity labeling with off-line avidin affinity fractionation of Cys-containing peptides (17,18). In this approach, 39 S subunits were treated with a reagent (ICAT TM DO reagent) which alkylates Cys residues with a tag carrying a biotin moiety.…”

Section: Resultsmentioning

confidence: 99%

“…The biotin tag on this reagent allowed purification of Cys-containing peptides using avidin affinity chromatography as described previously (17,18). For this analysis, 5 pmol of 39 S subunits was used for in situ biotinylation with the ICAT TM D0 reagent followed by digestion with trypsin as summarized in Fig.…”

Section: Preparation Of Bovine Mitochondrial Ribosomal Subunits-bovinementioning

confidence: 99%

See 3 more Smart Citations

The Large Subunit of the Mammalian Mitochondrial Ribosome

Koc¹,

Burkhart²,

Blackburn³

et al. 2001

Journal of Biological Chemistry

243

View full text Add to dashboard Cite

Identification of all the protein components of the large subunit (39 S) of the mammalian mitochondrial ribosome has been achieved by carrying out proteolytic digestions of whole 39 S subunits followed by analysis of the resultant peptides by liquid chromatography and mass spectrometry. Peptide sequence information was used to search the human EST data bases and complete coding sequences were assembled. The human mitochondrial 39 S subunit has 48 distinct proteins. Twenty eight of these are homologs of the Escherichia coli 50 S ribosomal proteins L1, L2, L3 , L4, L7/L12, L9, L10, L11, L13, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L24, L27, L28, L30, L32, L33, Mammalian mitochondria are responsible for the synthesis of 13 proteins localized in the inner membrane. These proteins are components of the oligomeric complexes essential for oxidative phosphorylation and, hence, for the synthesis of about 90% of the ATP in eukaryotic organisms. The 55 S mammalian mitochondrial ribosomes consists of small (28 S) and large (39 S) subunits (1). In contrast to bacterial ribosomes which are about 65% RNA, mammalian mitochondrial ribosomes are only 33% RNA. The low percentage of RNA in these ribosomes reflects a reduction in the size of the rRNA and a compensating increase in the number of ribosomal proteins. For example, the small subunit of the mammalian mitochondrial ribosome contains a 12 S rRNA (about 950 nucleotides) and an estimated 29 proteins (2). In contrast, the Escherichia coli 30 S subunit has a 16 S rRNA (1542 nucleotides in length) and 21 proteins (3). The large subunit of the mammalian mitochondrial ribosome contains a 16 S rRNA (about 1560 nucleotides) and about 50 proteins (4, 5).The identification of proteins in mammalian mitochondrial ribosomes has been challenging due to their low abundance. Recently 60 mammalian mitochondrial ribosomal proteins, 31 proteins from the large subunit and 29 proteins from the small subunit, have been characterized by different laboratories (2, 6 -14). The identification of these proteins used two approaches. The traditional approach was to separate the proteins on two-dimensional gels or high performance liquid chromatography followed by sequence analysis using Edman chemistry or mass spectrometry (MS). More recently, proteins present in the 28 S subunit have been characterized by proteolytic digestion of whole subunits. Sequence information on the peptides present in this complex mixture was obtained by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS).1 This strategy allowed the identification of 28 proteins of the small subunit including 14 proteins that had not previously been identified (2). In the present study, we have extended this approach to the 39 S subunit. In addition to direct analysis of 39 S digests by LC/MS, aliquots of the total digest were fractionated prior to reversed-phase LC/MS analysis to maximize the number of peptides sequenced. In the first approach, a portion of the total digest was fractionated by affinity selection ...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Preparation Of Bovine Mitochondrial Ribosomal Subunits-bovinementioning

confidence: 99%

See 2 more Smart Citations

The Large Subunit of the Mammalian Mitochondrial Ribosome

Koc¹,

Burkhart²,

Blackburn³

et al. 2001

Journal of Biological Chemistry

243

View full text Add to dashboard Cite

show abstract

“…This separation can be achieved via SDS polyacrylamide gel electrophoresis (SDS-PAGE) of the protein mixture followed by analysis of the individual slices [5] or by liquid chromatography-MS (LC-MS) analysis of the tryptic peptides derived from all proteins [6]. Using affinity tags introduced at specific amino acid residues and purification of peptides containing the affinity tag is a way to create a representative subset of only few tryptic peptides per original protein thereby increasing the numbers of proteins that can be detected in one MS experiment [7,8].…”

mentioning

confidence: 99%

Analysis of proteins copurifying with the cd4/lck complex using one-dimensional polyacrylamide gel electrophoresis and mass spectrometry: comparison with affinity-tag based protein detection and evaluation of different solubilization methods

Bernhard

Cunningham

Sheil

2004

J. Am. Soc. Mass Spectrom.

View full text Add to dashboard Cite

Mass spectrometry-based identification of the components of affinity purified protein complexes after polyacrylamide gel electrophoresis (PAGE) and in-gel digest has become very popular for the detection of novel protein interactions. As an alternative, the entire protein complex can be subjected to proteolytic cleavage followed by chromatographic separation of the peptides. Based on our earlier report of a method using affinity tag-mediated purification of cysteine-containing peptides to analyse proteins present in an affinity purification of the CD4/lck receptor complex, we here evaluated the use of one-dimensional polyacrylamide gel electrophoresis for analysis of the same receptor complex purification. Using electrospray and tandem mass spectrometry analyses of tryptic peptides from in-gel digested proteins we identified the components of the CD4 receptor complex along with 23 other proteins that were all likely to be non-specifically binding proteins and mainly different from the proteins detected in our previous study. We compare the alternative strategy with the affinity tag-based method that we described earlier and show that the PAGE-based method enables more proteins to be identified. We also evaluated the use of a more stringent lysis buffer for the CD4 purification to minimise non-specific binding and identified 52 proteins along with CD4 in three independent experiments suggesting that the choice of lysis buffer had no significant effect on the extent of non-specific binding. Non-specific binding was inconsistent and involved various types of proteins underlining the importance of reproducibility and control experiments in proteomic studies. (J Am Soc Mass Spectrom 2004, 15, 558 -567)

show abstract