Top-down protein identification of proteasome proteins with nanoLC-FT-ICR-MS employing data-independent fragmentation methods.

A comparison of different data-independent fragmentation methods combined with LC coupled to high-resolution FT-ICR-MS/MS is presented for top-down MS of protein mixtures. Proteins composing the 20S and 19S proteasome complexes and their PTMs were identified using a 15 T FT-ICR mass spectrometer. The data-independent fragmentation modes with LC timescales allowed for higher duty-cycle measurements that better suit online LC-FT-ICR-MS. Protein top-down dissociation was effected by funnel-skimmer collisionally activated dissociation (FS-CAD) and CASI (continuous accumulation of selected ions)-CAD. The N-termini for 9 of the 14 20S proteasome proteins were found to be modified, and the α3 protein was found to be phosphorylated; these results are consistent with previous reports. Mass-measurement accuracy with the LC-FT-ICR system for the 20- to 30-kDa 20S proteasome proteins was 1 ppm. The intact mass of the 100-kDa Rpn1 subunit from the 19S proteasome complex regulatory particle was measured with a deviation of 17 ppm. The CASI-CAD technique is a complementary tool for intact-protein fragmentation and is an effective addition to the growing inventory of dissociation methods that are compatible with online protein separation coupled to FT-ICR-MS.

Proteomic characterization of human milk fat globule membrane proteins during a 12 month lactation period.

The milk fat globule membrane (MFGM) contains proteins which have been implicated in a variety of health benefits. Milk fat globule membrane proteins were isolated from human milk during a 12 month lactation period and subjected to in-solution digestion and liquid chromatography tandem mass spectrometry analysis. Data were pooled, and our results showed that 191 proteins were identified. Relative quantification of the identified MFGM proteins during the course of lactation was performed by label free spectral counting and differentiation expression analysis, which showed some proteins decreasing during the course of lactation whereas some increased or remained at a relatively constant level. The human MFGM proteins are distributed between intracellular, extracellular, and membrane-associated proteins, and they are mainly involved in cell communication and signal transduction, immune function, metabolism and energy production. This study provides more insights into the dynamic composition of human MFGM proteins, which in turn will enhance our understanding of the physiological significance of MFGM proteins.

Proteomic characterization of human milk whey proteins during a twelve-month lactation period.

Human milk is a rich source of bioactive proteins that support the early growth and development of the newborn. Although the major components of the protein fraction in human milk have been studied, the expression and relative abundance of minor components have received limited attention. We examined the expression of low-abundance proteins in the whey fraction of human milk and their dynamic changes over a twelve-month lactation period. The low-abundance proteins were enriched by ProteoMiner beads, and protein identification was performed by liquid chromatography tandem mass spectrometry. One hundred and fifteen proteins were identified, thirty-eight of which have not been previously reported in human colostrum or milk. We also for the first time described differences in protein patterns among the low-abundance proteins during lactation. These results enhance our knowledge about the complexity of the human milk proteome, which constitutes part of the advantages to the breast-fed infant.

Proteomic characterization of specific minor proteins in the human milk casein fraction.

Human milk contains many bioactive proteins that are likely to support the early development of the newborn. The aim of this study was to identify whether there are specific minor proteins associated with the human milk casein micelle prepared by the acid precipitation method. Protein identification was performed by liquid chromatography tandem mass spectrometry analysis. Eighty-two proteins were identified in the casein micelle, 18 of which are not present in their whey compartment. Thirty-two of these proteins specifically associated with the casein micelle have not previously been identified in human milk or colostrum. Proteins involved in immune function comprised the major part (28%) of total proteins, and another significant part is involved in metabolism/energy production (22%). Most of the proteins were of extracellular or cytoplasmic origin (accounting for 50 and 29%, respectively). This study indicates that various soluble proteins should be considered as part of the casein compartment, prepared by the acid precipitation method. The data provide new insight not only into the proteomic profile of the human milk casein micelle and its physiological significance, but also into the proper proportion of casein and casein-associated proteins to use in infant formula.

Proteomic analysis of human nail plate.

Shotgun proteomic analysis of the human nail plate identified 144 proteins in samples from Causcasian volunteers. The 30 identified proteins solubilized by detergent and reducing agent, 90% of the total nail plate mass, were primarily keratins and keratin associated proteins. Keratins comprised a majority of the detergent-insoluble fraction as well, but numerous cytoplasmic, membrane, and junctional proteins and histones were also identified, indicating broad use by transglutaminases of available proteins as substrates for cross-linking. Two novel membrane proteins were identified, also found in the hair shaft, for which mRNAs were detected only at very low levels by real-time polymerase chain reaction in other tissues. Parallel analyses of nail samples from volunteers from Inner Mongolia, China gave essentially the same protein profiles. Comparison of the profiles of nail plate and hair shaft from the latter volunteers revealed extensive overlap of protein constituents. Analyses of samples from an arsenic-exposed population revealed few proteins whose levels were altered substantially but raised the possibility of detecting sensitive individuals in this way.

A comparative study of in-gel digestions using microwave and pressure-accelerated technologies.

One of the most popular methods to prepare tryptic peptides for bottom-up proteomic analysis is in-gel digestion. To date, there have been few studies comparing various digestion methods. In this study, we compare the efficiency of several popular in-gel digestion methods, along with new technologies that may improve digestion efficiency, using a human epidermoid carcinoma cell lysate protein standard. The efficiency of each protocol was based on the average number of proteins identified and their respective sequence coverage and relative quantitation using spectral counting. The importance of this study lies in its comparison of pre-existing in-gel digestion methods with those that use newly developed technologies that may introduce the potential for a more cost-effective digestion, higher protein yield, and an overall reduction in processing time. The following four protocols were compared: an overnight in-gel digestion protocol; an overnight in-gel digestion protocol, in which we remove the vacuum centrifugation steps; in-gel digestion in a barometric pressure cycler; and in-gel digestion in a scientific microwave. Several variables were tested for increased digestion efficiency and decreased keratin contamination. Statistical analysis was performed on replicate samples to determine significant differences between protocols.

In vivo multiplex quantitative analysis of 3 forms of alpha melanocyte stimulating hormone in pituitary of prolyl endopeptidase deficient mice.

BACKGROUND: In vitro reactions are useful to identify putative enzyme substrates, but in vivo validation is required to identify actual enzyme substrates that have biological meaning. To investigate in vivo effects of prolyl endopeptidase (PREP), a serine protease, on alpha melanocyte stimulating hormone (alpha-MSH), we developed a new mass spectrometry based technique to quantitate, in multiplex, the various forms of alpha-MSH. METHODS: Using Multiple Reaction Monitoring (MRM), we analyzed peptide transitions to quantify three different forms of alpha-MSH. Transitions were first confirmed using standard peptides. Samples were then analyzed by mass spectrometry using a triple quadrupole mass spectrometer, after elution from a reverse phase C18 column by a gradient of acetonitrile. RESULTS: We first demonstrate in vitro that PREP digests biological active alpha melanocyte stimulating hormone (alpha-MSH(1-13)), by cleaving the terminal amidated valine and releasing a truncated alpha melanocyte stimulating hormone (alpha-MSH(1-12)) product--the 12 residues alpha-MSH form. We then use the technique in vivo to analyze the MRM transitions of the three different forms of alpha-MSH: the deacetylated alpha-MSH(1-13), the acetylated alpha-MSH(1-13) and the truncated form alpha-MSH(1-12). For this experiment, we used a mouse model (PREP-GT) in which the serine protease, prolyl endopeptidase, is deficient due to a genetrap insertion. Here we report that the ratio between acetylated alpha-MSH(1-13) and alpha-MSH(1-12) is significantly increased (P-value = 0.015, N = 6) in the pituitaries of PREP-GT mice when compared to wild type littermates. In addition no significant changes were revealed in the relative level of alpha-MSH(1-13) versus the deacetylated alpha-MSH(1-13). These results combined with the demonstration that PREP digests alpha-MSH(1-13) in vitro, strongly suggest that alpha-MSH(1-13) is an in vivo substrate of PREP. CONCLUSION: The multiplex targeted quantitative peptidomics technique we present in this study will be decidedly useful to monitor several neuropeptide enzymatic reactions in vivo under varying conditions.