Miscellaneous
 

Other proteins

A selection of other proteins retrieved in when analysing the histone samples by
LC-MS/MS analysis.

Protein name
Protein component of the small (40S) ribosomal subunit; has similarity to E. coli S15 and rat S13 r
Rpl27bp: 60S ribosomal protein L27, identical to Yhr010p from GenBank Accession Number U10400; CAI:
Ribosomal protein L29 of the large (60S) ribosomal subunit, has similarity to E. coli L15 and rat L
Tef1p: Elongation factor 1-alpha [Saccharomyces cerevisiae]
Histone variant H2AZ, exchanged for histone H2A in nucleosomes by the SWR1 complex;
N-terminally acetylated protein component of the large (60S) ribosomal subunit, nearly identical to
Protein component of the small (40S) ribosomal subunit; nearly identical to Rps18Bp and has similar
Protein component of the large (60S) ribosomal subunit, nearly identical to Rpl14Ap and has similar
Protein component of the small (40S) ribosomal subunit; nearly identical to Rps22Bp and has similar
Ribosomal protein 51 (rp51) of the small (40s) subunit; nearly identical to Rps17Ap and has similar
Eft2p [Saccharomyces cerevisiae]
Srp40p [Saccharomyces cerevisiae]
Protein component of the small (40S) ribosomal subunit; identical to Rps24Bp and has similarity to
Ribosomal protein 59 (rp59) of the small (40S) ribosomal subunit, required for ribosome assembly; m
Major of three pyruvate decarboxylase isozymes, key enzyme in alcoholic fermentation, decarboxylate
Protein compoent of the large (60S) ribosomal sub-style:solid; border-right-width:1; border-top-style:none; border-top-width:medium; border-bottom-style:none; border-bottom-width:medium"> Protein component of the large (60S) ribosomal subunit, has similarity to rat L30 ribosomal protein
Protein component of the large (60S) ribosomal subunit, nearly identical to Rpl13Bp; not essential
Protein component of the large (60S) ribosomal subunit, has similairty to rat L32 ribosomal protein
Ribosomal protein 28 (rp28) of the small (40S) ribosomal subunit, required for translational accura
Protein component of the small (40S) ribosomal subunit; identical to Rps16Ap and has similarity to
calmodulin [Saccharomyces cerevisiae]

 

Selecting peptides for fragmentation (MS/MS)

The mass spectrometers used for the LC-MS/MS analysis's, automatics select ions for
MS/MS experiments. Criteria's used in the selections are 1) abundance and 2) charge
state of a ion.
With regard to abundance: using LC in combination with MS results in an highly dynamic
situation. Every peptide is only available for a short time period (typical 20 sec.)
and depending on the complexity of an sample and the nature of the peptides to be analyzed,
multiple peptide ions can elute simultaneously.
The mass spectrometer cycle between a MS survey scan, where ions are evaluated and
hereafter selected or rejected for MS/MS experiments, using the above criteria's. 
Followed the survey scan, the mass spectrometer is shifted to MS/MS operation and the
selected ions are subjected to MS/MS experiments. After a number of MS/MS experiments,
a new survey scan is conducted, and a new set of ions are selected for MS/MS
experiments. As follow of the dynamic nature of the experiments, ions will be missed
- most often ions of lower abundance. As also follows, if a given peptide has not been
selected for MS/MS experiments, it will not be identified, but it can not be concluded
that the peptide is not present in the sample.

 

Fig. 1. Example of a cycle with one survey MS scan where three ions are selected fragmentation.

 

Quantitation, using mass spectrometry

Many considaratons are needed. The two main things to remember are: 1) two different
peptides from the same protein - and also the same peptide with different modifications
- are likely to behave different, meaning, intensities can not be compared. 2) It is
difficult to compare abundances of exactely the same peptide found in two different
samples. Measures can be taken to make it easier, but many pre-cautions needs to be
taken - and present, I believe that is is difficult to have the results accepted in
peer reviewed journals without doing a experiment using internal relative standards.

The answers to the above is the use of either a standard (a isotopically labeled
peptide), in-vivo or in-vitro isotopic labeling of the samples. A recent example of
the use of a isotopical labeled standard where presented by Alain Verreault. In-vitro
labeling are exemplified by DeSouza et al. [J Proteome Res. 2005, p377], and in-vivo,
by Ibarrola et al. [Analytical Chemistry, 2003, p6043]

 

Fragment marker ions for Lysine modifications (acetylation/methylation)

This table summarizes work presented by Zhang et al.[Proteomics, 2004, p1].

Modification Fragment marker ion [m/z] Neutral loss [Da]
K (not modified) 84  
K (single methylation) 84 & 98  
K (di-methylation) 84  
K (tri-methylation) 84 59
K (Acetylation) 84 & 126