While it is generally recognized that misfolding of specific proteins can cause late-onset disease, the contribution of protein aggregation to the normal aging process is less well understood. To address this issue, a mass spectrometry-based proteomic analysis was performed to identify proteins that adopt sodium dodecyl sulfate (SDS)-insoluble conformations during aging in Caenorhabditis elegans. SDS-insoluble proteins extracted from young and aged C. elegans were chemically labeled by isobaric tagging for relative and absolute quantification (iTRAQ) and identified by liquid chromatography and mass spectrometry. Two hundred and three proteins were identified as being significantly enriched in an SDS-insoluble fraction in aged nematodes and were largely absent from a similar protein fraction in young nematodes. The SDS-insoluble fraction in aged animals contains a diverse range of proteins including a large number of ribosomal proteins. Gene ontology analysis revealed highly significant enrichments for energy production and translation functions. Expression of genes encoding insoluble proteins observed in aged nematodes was knocked down using RNAi, and effects on lifespan were measured. 41% of genes tested were shown to extend lifespan after RNAi treatment, compared with 18% in a control group of genes. These data indicate that genes encoding proteins that become insoluble with age are enriched for modifiers of lifespan. This demonstrates that proteomic approaches can be used to identify genes that modify lifespan. Finally, these observations indicate that the accumulation of insoluble proteins with diverse functions may be a general feature of aging.

SUMMARY Reducing protein synthesis slows growth and development but can increase adult life span. We demonstrate that knockdown of eukaryotic translation initiation factor 4G (eIF4G), which is downregulated during starvation and dauer state, results in differential translation of genes important for growth and longevity in C. elegans. Genome-wide mRNA translation state analysis showed that inhibition of IFG-1, the C. elegans ortholog of eIF4G, results in a relative increase in ribosomal loading and translation of stress response genes. Some of these genes are required for life span extension when IFG-1 is inhibited. Furthermore, enhanced ribosomal loading of certain mRNAs upon IFG-1 inhibition was correlated with increased mRNA length. This association was supported by changes in the proteome assayed via quantitative mass spectrometry. Our results suggest that IFG-1 mediates the antagonistic effects on growth and somatic maintenance by regulating mRNA translation of particular mRNAs based, in part, on transcript length.

The rapid chemical analysis of individual cells is an analytical capability that will profoundly impact many fields including bioaerosol detection for biodefense and cellular diagnostics for clinical medicine. This article describes a mass spectrometry-based analytical technique for the real-time and reagentless characterization of individual airborne cells without sample preparation. We characterize the mass spectral signature of individual Bacillus spores and demonstrate the ability to distinguish two Bacillus spore species, B. thuringiensis and B.atrophaeus, from one another very accurately and from the other biological and nonbiological background materials tested with no false positives at a sensitivity of 92%. This example demonstrates that the chemical differences between these two Bacillus spore species are consistently and easily detected within single cells in seconds.

Bioaerosol mass spectrometry is being developed to analyze and identify biological aerosols in real time. Characteristic mass spectra from individual bacterial endospores of Bacillus subtilis var. niger were obtained in a bipolar aerosol time-of-flight mass spectrometer using a pulsed 266-nm laser for molecular desorption and ionization. Spectra from single spores collected at an average fluence of approximately 0.1 J/cm2 frequently contain prominent peaks attributed to arginine, dipicolinic acid, and glutamic acid, but the shot-to-shot (spore-to-spore) variability in the data may make it difficult to consistently distinguish closely related Bacillus species with an automated routine. Fortunately, a study of the laser power dependence of the mass spectra reveals clear trends and a finite number of "spectral types" that span most of the variability. This, we will show, indicates that a significant fraction of the variability must be attributed to fluence variations in the profile of the laser beam.

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BackgroundMuscle mass can be measured directly in vivo by isotope dilution, using Creatine‐(methyl‐d3) monohydrate (D3‐Cr) by mouth followed by measurement of the steady‐state enrichment of D3‐creatinine (D3‐Crn) in urine. Isotope dilution methods require knowledge of the amount of tracer delivered to the pool of interest. In a subset of human subjects, a small amount of orally administered D3‐Cr ‘spills’ into urine after absorption and prior to transport into skeletal muscle cells. The objectives were to develop a method to correct for spillage to compare the estimate of muscle mass by D3‐Cr dilution to other assessments of fat‐free mass.MethodsSubjects (19 males, 23–81 years old; 20 females, 20–77 years old) ingested a single dose of 60 mg D3‐Cr and urine was collected prior to and daily for 4 days following the dose. Fasting morning urine samples was assessed for D3‐Cr, total Cr, D3‐Crn, and total Crn concentrations, as well as isotopic enrichments of D3‐Crn, by LC/MS. The 24‐h urine collections over 3 days after the dose of D3‐Cr were also performed to determine D3‐Cr spillage. Total body water, fat mass, and fat‐free mass were assessed by bioelectrical impedance spectroscopy (BIS).ResultsSpillage of D3‐Cr in the urine was greater in women than men. D3‐Crn enrichment and the ratio of Cr/Crn were used in an algorithm to calculate Cr pool size and muscle mass. Specifically, an algorithm was developed for the estimation of spillage based on the relationship between the fasting Cr/Crn ratio and the cumulative proportion of the D3‐Cr dose excreted over 3 days based on 24‐h urine collections. Muscle mass corrected using the algorithm based on fasting urine levels correlated (r = 0.9967, P < 0.0001) with that corrected by measuring D3‐Cr dose excreted. Muscle mass measured by D3‐Crn enrichment also correlated (r = 0.8579, P < 0.0001, algorithm corrected) with that measured by 24‐h Crn excretion. Muscle mass measured by D3‐Cr dilution method correlated with intracellular water by BIS, whether using spillage corrected by the algorithm (r = 0.9041, P < 0.0001) or measured by 3 day D3‐Cr losses (r = 0.91, P < 0.0001) and similarly correlated with fat‐free mass by BIA (r = 0.8857 and 0.8929, P < 0.0001, respectively).ConclusionsThe D3‐Cr dilution method is further validated here as a non‐invasive, easy‐to‐use test for measuring muscle mass. The technical issue of D3‐Cr spillage can be corrected for with a simple algorithm based on fasting spot urine samples. Muscle mass by Cr dilution potentially has broad applications in clinical and research settings.

Single vegetative cells and spores of Bacillus atrophaeus, formerly Bacillus subtilis var. niger, were analyzed using bioaerosol mass spectrometry. Key biomarkers were identified from organisms grown in 13C and 15N isotopically enriched media. Spore spectra contain peaks from dicipolinate and amino acids. The results indicate that compounds observed in the spectra correspond to material from the spore's core and not the exosporium. Standard compounds and mixtures were analyzed for comparison. The biomarkers for vegetative cells were clearly different from those of the spores, consisting mainly of phosphate clusters and amino acid fragments.

Molecular markers associated with CNS injury are of diagnostic interest. Mechanical trauma generates cellular deformation associated with membrane permeability with unknown molecular consequences. We used an in vitro model of stretch-injury and proteomic analyses to determine protein changes in murine astrocytes and their surrounding fluids. Abrupt pressure-pulse stretching resulted in the rapid release of 59 astrocytic proteins with profiles reflecting cell injury and cell death, i.e. mechanoporation and cell lysis. This acute trauma-release proteome was overrepresented with metabolic proteins compared to the uninjured cellular proteome, bearing relevance for post-traumatic metabolic depression. Astrocyte-specific deletion of signal transducer and activator of transcription 3 (STAT3-CKO) resulted in reduced stretch-injury tolerance, elevated necrosis and increased protein release. Consistent with more lysed cells, more protein complexes, nuclear and transport proteins were released from STAT3-CKO versus non-transgenic astrocytes. STAT3-CKO astrocytes had reduced basal expression of GFAP, lactate dehydrogenase B (LDHB), aldolase C (ALDOC) and astrocytic phosphoprotein 15 (PEA15), and elevated levels of tropomyosin (TPM4) and α actinin 4 (ACTN4). Stretching caused STAT3 dependent cellular depletion of PEA15 and GFAP, and its filament disassembly in subpopulations of injured astrocytes. PEA15 and ALDOC signals were low in injured astrocytes acutely after mouse spinal cord crush injury and robustly expressed in reactive astrocytes one day post-injury. In contrast, α crystallin (CRYAB) was present in acutely injured astrocytes, and absent from uninjured and reactive astrocytes, demonstrating novel marker differences among post-injury astrocytes. These findings reveal a proteomic signature of traumatically-injured astrocytes reflecting STAT3-dependent cellular survival with potential diagnostic value.