25 research outputs found
Improved Reversed Phase Chromatography of Hydrophilic Peptides from Spatial and Temporal Changes in Column Temperature
Identification of the Components of Simple Protein Mixtures by High-Accuracy Peptide Mass Mapping and Database Searching
Improved Reversed Phase Chromatography of Hydrophilic Peptides from Spatial and Temporal Changes in Column Temperature
Reversed
phase chromatography is an established method for peptide
separation and frequently coupled to electrospray ionization–mass
spectrometry for proteomic analysis. Column temperature is one parameter
that influences peptide retention and elution, but it is often overlooked
as its implementation requires additional equipment and method optimization.
An apparatus that allows temperature manipulation in three areas of
a two-column setup was evaluated for improvements in chromatography.
Using commercially available standards, we demonstrate that a low
column temperature (0 °C) during sample loading enhances the
peak shape of several bovine serum albumin hydrophilic peptides. For
digested HeLa lysates, approximately 15% more peptide identifications
were obtained by increasing the precolumn temperature to 50 °C
after the 500 ng sample was loaded at a low temperature. This method
also identified additional early eluting peptides with grand average
of hydropathicity values less than −2. We also investigated
the effect of cooler column temperatures on peptides with post-translational
modifications. It was possible to minimize the coelution of an isoaspartylated
peptide and its unmodified version when the analytical column temperature
was decreased to 5 °C. Aside from demonstrating the utility of
lower temperatures for improved chromatography, its application at
specific locations and time points is critical for peptide detection
and separation
Yeast poly(A)-binding protein Pab1 shuttles between the nucleus and the cytoplasm and functions in mRNA export
Pab1 is the major poly(A)-binding protein in yeast. It is a multifunctional protein that mediates many cellular functions associated with the 3′-poly(A)-tail of messenger RNAs. Here, we characterize Pab1 as an export cargo of the protein export factor Xpo1/Crm1. Pab1 is a major Xpo1/Crm1-interacting protein in yeast extracts and binds directly to Xpo1/Crm1 in a RanGTP-dependent manner. Pab1 shuttles rapidly between the nucleus and the cytoplasm and partially accumulates in the nucleus when the function of Xpo1/Crm1 is inhibited. However, Pab1 can also be exported by an alternative pathway, which is dependent on the MEX67-mRNA export pathway. Import of Pab1 is mediated by the import receptor Kap108/Sxm1 through a nuclear localization signal in its fourth RNA-binding domain. Interestingly, inhibition of Pab1’s nuclear import causes a kinetic delay in the export of mRNA. Furthermore, the inviability of a pab1 deletion strain is suppressed by a mutation in the 5′–3′ exoribonuclease RRP6, a component of the nuclear exosome. Therefore, nuclear Pab1 may be required for efficient mRNA export and may function in the quality control of mRNA in the nucleus
Phosphopeptide detection and sequencing by matrix-assisted laser desorption/ionization quadrupole time-of-flight tandem mass spectrometry.
Rrp47p Is an Exosome-Associated Protein Required for the 3′ Processing of Stable RNAs
Related exosome complexes of 3′→5′ exonucleases are present in the nucleus and the cytoplasm. Purification of exosome complexes from whole-cell lysates identified a Mg(2+)-labile factor present in substoichiometric amounts. This protein was identified as the nuclear protein Yhr081p, the homologue of human C1D, which we have designated Rrp47p (for rRNA processing). Immunoprecipitation of epitope-tagged Rrp47p confirmed its interaction with the exosome and revealed its association with Rrp6p, a 3′→5′ exonuclease specific to the nuclear exosome fraction. Northern analyses demonstrated that Rrp47p is required for the exosome-dependent processing of rRNA and small nucleolar RNA (snoRNA) precursors. Rrp47p also participates in the 3′ processing of U4 and U5 small nuclear RNAs (snRNAs). The defects in the processing of stable RNAs seen in rrp47-Δ strains closely resemble those of strains lacking Rrp6p. In contrast, Rrp47p is not required for the Rrp6p-dependent degradation of 3′-extended nuclear pre-mRNAs or the cytoplasmic 3′→5′ mRNA decay pathway. We propose that Rrp47p functions as a substrate-specific nuclear cofactor for exosome activity in the processing of stable RNAs